Methods and compositions for expressing polynucleotides specifically in smooth muscle cells in vivo

ABSTRACT

The present invention generally relates to promoters, enhancers and other regulatory elements of smooth muscle cells (“SMC”). The invention more particularly relates to methods for the targeted knockout, or over-expression, of genes of interest within smooth muscle cells or within a subtype of smooth muscle cells. The invention further relates to methods of conferring polynucleotide expression in vivo specifically in smooth muscle cells or in subtypes of smooth muscle cells.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 60/263,811, filed Jan. 24, 2001. The present application is also acontinuation-in-part application of U.S. patent application Ser. No.09/600,319, filed Jul. 13, 2000, now U.S. Pat. No. 6,780,610, which is aU.S. national phase application of international application numberPCT/US99/01038, filed Jan. 15, 1999, which claims priority to U.S.Provisional Application No. 60/071,300, filed Jan. 16, 1998. Theinvention is also related to international application PCT/US99/24972.Each of the aforementioned applications is explicitly incorporatedherein by reference in its entirety and for all purposes.

STATEMENT AS TO FEDERAL FUNDING

Work described herein has been supported in part by National Institutesof Health grants R01 HL38854 and P01 HL19242. The U.S. Government maytherefore have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the field of regulation of geneexpression, and specifically to smooth muscle specific promoters andenhancers. The invention also relates to methods of modulating geneexpression by utilizing smooth muscle specific promoters and enhancers.

BACKGROUND OF THE INVENTION

Smooth muscle cells (SMCs), often termed the most primitive type ofmuscle cell because they most resemble non-muscle cells, are called“smooth” because they contain no striations, unlike skeletal and cardiacmuscle cells. Smooth muscle cells aggregate to form smooth muscle whichconstitutes the contractile portion of the stomach, intestine anduterus, the walls of arteries, the ducts of secretory glands and manyother regions in which slow and sustained contractions are needed.

Abnormal gene expression in SMC plays a major role in numerous diseasesincluding, but not limited to, atherosclerosis, hypertension, stroke,asthma and multiple gastrointestinal, urogenital and reproductivedisorders. These diseases are the leading causes of morbidity andmortality in Western Societies, and account for billions of dollars inhealth care costs in the United States alone each year.

In recent years, the understanding of muscle differentiation has beenenhanced greatly with the identification of several key cis-elements andtrans-factors that regulate expression of muscle-specific genes. FirulliA. B., et al., 1997, Trends in Genetics, 13:364-369; Sartorelli V. etal., 1993, Circ. Res., 72:925-931. However, the elucidation oftranscriptional pathways that govern muscle differentiation has beenrestricted primarily to skeletal and cardiac muscle. Currently, notranscription factors have yet been identified that direct smoothmuscle-specific gene expression, or SMC myogenesis. Owens G. K., 1995,Physiol. Rev., 75:487-517. Unlike skeletal and cardiac myocytes, SMC donot undergo terminal differentiation. Furthermore, they exhibit a highdegree of phenotype plasticity, both in culture and in vivo. Owens G.K., 1995, Physiol. Rev., 75:487-517; Schwartz, S. M. et al., 1990,Physiol. Rev., 70:1177-1209. Phenotype plasticity is particularlystriking when SMC located in the media of normal vessels are compared toSMC located in intimal lesions resulting from vascular injury oratherosclerotic disease. Schwartz, S. M. et al., 1990, Physiol. Rev.,70:1177-1209; Ross R., 1993, Nature, 362:801-809; Kocher O. et al.,1991, Lab. Invest., 65:459-470; Kocher O. et al., 1986, Hum. Pathol.,17:875-880. Major modifications include decreased expression of smoothmuscle isoforms of contractile proteins, altered growth regulatoryproperties, increased matrix production, abnormal lipid metabolism anddecreased contractility. Owens G. K., 1995, Physiol. Rev., 75:487-517.The process by which SMC undergo such changes is referred to as“phenotypic modulation”. Chamley-Campbell J. H. et al., 1981,Atherosclerosis, 40:347-357. Importantly, these alterations inexpression patterns of SMC protein cannot simply be viewed as aconsequence of vascular disease, but rather are likely to contribute tothe progression of the disease.

Expression of smooth muscle myosin heavy chain (SM-MHC) appears to becompletely restricted to SMC lineages throughout development (Miano J.et al., 1994, Circ. Res., 75:803-812). To date, four SM-MHC isoforms(SMC-1A, SMC-1B, SMC-2A, and SMC-2B) have been identified (Nagai R. etal., 1989, J. Biol. Chem., 264:9734-9737; White S. et al., 1993, Am. J.Physiol., 264:C1252-C1258; Kelley C. A. et al., 1993, J. Biol. Chem.,268:12848-12854), all of which are derived from alternative splicing ofa single gene (Miano J. et al., 1994. Circ. Res., 75:803-812; Babij P.et al., 1989, J. Mol. Biol., 210:673-679). Alterations in expression ofSM-MHC isoforms have been extensively documented in SMC that haveundergone phenotypic modulation either when placed in culture (Rovner A.S., 1986, J. Biol. Chem., 261:14740-14745; Kawamoto S. et al., 1987, J.Biol. Chem., 262:7282-7288), or in vascular lesions of both humans andseveral animal models of vascular disease (Aikawa M. et al., 1997,Circulation, 96:82-90; Sartore S, et al., 1994, J. Vasc. Res.,31:61-81).

Transcriptional regulation of the SM-MHC gene has been analyzed incultured SMC and several functional cis-elements have been identified.White S. L. et al., 1996, J. Biol. Chem., 271:15008-15017; Katoh Y. etal., 1994, J. Biol. Chem., 269:30538-30545; Watanabe M. et al., 1996,Circ. Res., 78:978-989; Kallmeier R. C. et al., 1995, J. Biol. Chem.,270:30949-30957; Madsen C. S. et al., 1997, J. Biol. Chem.,272:6332-6340; Madsen C. S. et al., 1997, J. Biol Chem.,272:29842-29851. However, because differentiation of SMC is known to bedependent on many local environmental cues that cannot be completelyreproduced in vitro, cultured SMC are known to be phenotypicallymodified as compared to their in vivo counterparts (Owens G. K., 1995,Physiol. Rev., 75:487-517; Chamley-Campbell J. H. et al., 1981,Atherosclerosis, 40:347-357). As such, certain limitations may applyregarding the usefulness of cultured SMC in defining transcriptionalprograms that occur during normal SMC differentiation and maturationwithin the animal.

A few promoters relating to smooth muscles have been described in theart, e.g., promoters for SM-actin and SM22 genes. However, a majordisadvantage with these promoters is that they are clearly not SMCspecific. SM22 and SM-actin are highly expressed in myofibroblastsduring wound repair, within granulomatous tissues, tumors, etc. Thepromoters for these genes are also transiently activated in skeletal andcardiac muscle during development, and in association with a number ofpathological circumstances (e.g. myocardial hypertrophy). In addition,the SM22 promoter fragments tested to date also have very littleactivity in SMC tissues of adult mice. Thus, such promoters have majorlimitations in terms of their utility in smooth muscle tissue specifictargeting and expression in vivo.

Thus, there is a need in the art for transcription regulatory sequences(e.g., promoters and enhancers) that can direct gene expressionspecifically in smooth muscle tissues in vivo (e.g., in human ornon-human animals). There is also a need for relatively small smoothmuscle specific promoter/enhancers that retain high level SMC specificexpression in vivo and yet are selectively active in subsets of SMC(e.g. vascular versus gastrointestinal SMC, large versus small arteries,pulmonary versus gastrointestinal SMC, etc.). Methods for utilizing suchSMC specific promoters and enhancers to target delivery and expressionof polynucleotide to SMCs are also needed. The present inventionfulfills these and other needs.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides isolated or recombinantpolynucleotides which comprise a smooth muscle myosin heavy chain(SM-MHC) promoter/enhancer sequence capable of conferring smooth musclespecific expression in vivo. In some of the polynucleotides, thepromoter sequence consists essentially of a sequence selected from (i)the region of nucleotides 5663 to 5889 of SEQ ID NO:16; (ii) SEQ IDNO:16 except that CArG2 has been mutated; (iii) SEQ ID NO:16 except thatthe intronic CArG has been mutated; (iv) the regions of nucleotides 1 to6,700 and nucleotides 9,500 to 15,800 of SEQ ID NO:16; (v) the regionsof nucleotides 1 to 9,500 and nucleotides 11,700 to 13,700 of SEQ IDNO:16; (vi) SEQ ID NO:16; and (vii) SEQ ID NO:17.

Some of the polynucleotides hybridize under stringent conditions to theSM-MHC promoter/enhancer. Some of the polynucleotides further comprise aheterologous polynucleotide operably linked to the SM-MHC promotersequence. Some of the heterologous polynucleotides encode a polypeptide.The polypeptide can be a toxin, a prodrug-converting enzyme, a tumorsuppressor, a sensitizing agent, an apoptotic factor, an angiogenesisinhibitor, a cytokine, or an immunogenic antigen. Some of theheterologous polynucleotides consist of an antisense polynucleotide or acatalytic polynucleotide.

In another aspect, the invention provides expression vectors whichcomprise a smooth muscle myosin heavy chain (SM-MHC) promoter/enhancersequence that confers smooth muscle specific expression in vivo. Some ofthe expression vectors are retroviral vectors, adeno-associated viralvectors, or adenoviral vectors. Some of the expression vectors have thepromoter sequence operably linked to a heterologous polynucleotide. Someof the expression vectors comprise a promoter which consists essentiallyof the sequence of SEQ ID NO:16 except that CArG2 or the intronic CArGhas been mutated.

In another aspect, the invention provides genetically engineered hostcells comprising an expression vector of the invention. Transgenicnon-human animals containing the polynucleotides of the invention arealso provided. The invention also provides pharmaceutical compositionswhich comprise the polynucleotides of the invention in apharmaceutically acceptable carrier.

In still another aspect, the present invention provides methods ofexpression a polynucleotide in a smooth muscle cell in vivo. The methodsentail introducing into the smooth muscle cell the polynucleotide thatis operably linked to an SM-MHC promoter/enhancer sequence capable ofconferring smooth muscle specific expression in vivo. In some of themethods, the promoter/enhancer consists essentially of (i) the region ofnucleotides 5663 to 5889 of SEQ ID NO:16; (ii) SEQ ID NO:16 except thatCArG2 has been mutated; (iii) SEQ ID NO:16 except that the intronic CArGhas been mutated; (iv) the regions of nucleotides 1 to 6,700 andnucleotides 9,500 to 15,800 of SEQ ID NO:16; (v) the regions ofnucleotides 1 to 9,500 and nucleotides 11,700 to 13,700 of SEQ ID NO:16;(vi) SEQ ID NO:16; or (vii) SEQ ID NO:17.

In some methods, the polynucleotide to be expressed is a reporter gene.In some other methods, the polynucleotide to be expressed encodes atherapeutic protein. In some methods, the SM-MHC promoter/enhancerenables expression of the polynucleotide specifically in coronaryartery, aorta, airway smooth muscle, or pulmonary vascular smoothmuscle. In some methods, the SM-MHC promoter/enhancer enables expressionof the polynucleotide specifically in bladder smooth muscle,gastrointestinal tract smooth muscle, or urinary tract smooth muscle. Insome other methods, the SM-MHC promoter/enhancer enables expression ofthe polynucleotide specifically in aorta, pulmonary airway, or pulmonaryvascular smooth muscle. In still some other methods, the SM-MHCpromoter/enhancer enables expression of the polynucleotide specificallyin gastrointestinal tract smooth muscle, urinary tract smooth muscle,airway smooth muscle, vein smooth muscle, or small branching arterysmooth muscle. In some methods, the SM-MHC promoter/enhancer enablesexpression of the polynucleotide specifically in aorta artery smoothmuscle, carotid artery smooth muscle, pulmonary artery smooth muscle,vena cava vein smooth muscle, or vascular smooth muscle.

In yet another respect, the invention provides methods for screeningcompounds that modulate the activity of an SM-MHC promoter/enhancer. Themethods entail contacting a test compound with a cell that contains theSM-MHC promoter/enhancer operably linked to a reporter gene; detectingexpression of the reporter gene; and comparing the expression thusdetected with the amount of expression obtained in the absence of thetest compound. If the level obtained in the presence of the testcompound is higher or lower than that obtained in the absence of thetest compound, a compound that modulates the activity of the SM-MHCpromoter/enhancer has been identified.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification, the figures and claims.

All publications, GenBank deposited sequences, ATCC deposits, patentsand patent applications cited herein are hereby expressly incorporatedby reference in their entirety and for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression of the rat SM-MHC −4.2 to +11.6 promoter-lacZgene in vivo in adult transgenic mice showing the SMC specificity of thepromoter. Extremely high expression was observed in virtually all SMCtissues with no expression in non-SMC (FIG. 3).

FIG. 2 shows analysis of the SMC specificity of the rat SM-MHC promoterin various SMC tissues of transgenic mice in vivo using a crerecombinase indicator system. Transgenic mice carrying a SM MHC-crerecombinase gene were crossed to an indicator line containing a lox p(the cre recognition site) flanked stop codon inserted upstream of alacZ reporter gene that was inserted into the unbiquitiously expressedROSA gene locus by homologous recombination (the mouse is designatedR26R). Results showed expression of the lacZ indicator gene in virtuallyall SMC tissues. These results thus provide extremely compellingevidence for the SMC specificity of the −4.2 to +11.6 SM MHC promoter,since this assay system is far more sensitive in detecting reporterexpression than conventional direct reporter systems in that eventransient activation of the promoter is detected. That is, once crerecombination occurs there is permanent activation of the lacZ reporter.These results also establish the feasibility of using the SM MHCpromoter in conjunction with cre recombinase and conditional (e.g.tetracylcine etc.) gene regulatory systems for purposes of achieving SMCspecific gene targeting that is regulatable. A1-Thoracic aorta from aSMMHC-cre X R26R mouse; B-Trachea and carotid arteries; C-Heart; D-Lung;E-Skeletal muscle arteriole; F-Mesenteric vessels; G-Ventral surface ofcerebrum; H1-Jejunum from a SMMHC-cre X R26R mouse; I-Bladder. Theresults provide rigorous assessment of the complete SMC specificity ofthe SM MHC promoter.

FIG. 3 shows histological assessment of LacZ expression in the SMCtissues shown in FIG. 2 showing the remarkable SMC specificity ofexpression of the −4.2 to +11.6 SM-MHC promoter. The results showedcomplete specificity of expression of LacZ within SMC with the exceptionof a very small population of atrial myocytes that show transientactivation of the promoter during early heart formation (see reference{6812} f). A1-cross section of carotid artery; A2-cross section ofaorta; B1-cross section of intramyocardial artery; B2-staining of asmall population of cardiac myocytes; C-cross section of trachea;D-cross section of lung showing both bronchiole and pulmonary arterystaining; E-skeletal muscle arteriole and venule; F-cross section ofjejunum; G-cross section of esophagus; H-cross section of ureter.

FIG. 4 shows expression of the rat SM-MHC −4.2 to +5.3/+7.5 to +9promoter LacZ gene in various tissues from adult transgenic mice. Asseen, high reporter expression was seen in multiple SMC tissuesincluding the coronary arteries, aorta, airway SMC, and pulmonaryvascular SMC (PA-pulmonary artery). The results indicate that thisderivative of the SM MHC promoter retains high activity in aortic SMC,pulmonary arterial SMC, and airway SMC.

FIG. 5 shows histological section of the −4.2 to +5.3/+7.5 to +9 SM-MHCpromoter showing high specificity of expression in pulmonary arteriesand arterioles (see arrow).

FIG. 6 shows expression of the −4.2 to +2.5 and +5.3 to +11.6 SM-MHCLacZ gene in tissues of adult transgenic mice. As seen, this SM MHCpromoter reporter construct retained high level expression in thepulmonary airways, and aorta but diminished expression in the coronaryarteries as compared to the wildtype −4.2 to +11.6 SM MHC LacZ transgeneconstruct (see FIGS. 1-3). There was also high level expression inpulmonary vascular SMC based on histological analyses (data not shown).The results indicate high activity in pulmonary artery SMC, airway SMC,and the aorta, but virtually no activity in coronary artery SMC.

FIG. 7 shows transgene expression of the intronic CArG region-minimalTK-LacZ. Various tissues of 4-week-old transgenic mice and embryos ofthe 3xICR-TK LacZ line (7240) were stained for β-galactosidase activity.A-B: anterior view of the heart and lung; C: the esophagus, stomach, andduodenum; D: a part of small intestine; E: the bladder; F: bottom viewof the brain; G: anterior view of abdominal organs and great bloodvessels; H-K: histological examination of the thoracic aorta (H),pulmonary artery and bronchus (I), cardiac muscle and coronary artery(J), and intercostal muscle (K) of the 3xICR-TK LacZ transgenic mice;L-M: transgene expression in a 19.5-dpc embryo of the 3xICR-TK LacZline. The embryo was skinned, sectioned sagittally along the midline,stained, and cleared; N: transgene expression in the heart and aorta ofa 16.5-dpc embryo. Ao indicates aorta; PA, pulmonary artery; SMA,superior mesenteric artery; IVC, inferior vena cava; H, heart; Br,bronchus; Eso, esophagus; Int, intestine; S, stomach; B1, bladder. Theresults indicate that a very small derivative of the promoter is capableof driving high level expression in SMC in vivo.

FIG. 8 shows the effects of mutation of the intronic CArG on expressionof the rat −4.2 to +11.6 SM-MHC transgene in vivo. Abdominal organs wereremoved en block showing reporter expression in the blood vessels andurinary tract in the wild-type (A) and intronic CArG mutant (B)transgenic mice. To better illustrate transgene expression in largearteries, several smaller arteries and connective tissues were removedand the tissues cleared. The supramesenteric artery, which was stainedpositive, was removed from the intronic CArG mutant mouse tissues. Aportion of the tissues is expanded in the insert in panel B. Arrowheadsindicate the position of aorta that is not visible because of the lackof staining. Note that the blood vessels within the kidneys were notstained in either wild-type nor intronic CArG mutants. C, D, thethoracic aorta and branching arteries of the wild-type (C) and theintronic CArG (D) mutant transgenic mice. E, view of the large arteriesin the cervicothoracic region of the intronic CArG mutant transgenicmouse. F, the large arteries and their branches in the abdomen of theintronic CArG mutant. A portion of the arteries is expanded in theinsert in F. G, H, I, J, histological examination of the abdominal aortaand inferior vena cava of the wild-type (G, I) and the intronic CArGmutant (H, J) mice showing abrogation of reporter expression in SMCs ofthe aorta in the intronic CArG mutants. Note that expression in the venacava was not changed by the mutation. The boxed areas (G, H) are shownby a higher magnification (I, J). Ao indicates aorta; DA, ductusarteriosus; IVC, inferior vena cava; SCA, subclavian artery. The resultsindicate that this mutation selectively abolished activity in largeblood vessels such as the aorta, carotid, and coronary outflow tractswithout altering expression in smaller arteries and arterioles.

FIG. 9 shows activity of human SM-MHC promoter of −5.1 to +13.5 regionin transgenic mice. Expression of the human MHC−5.1/13.5-LacZ transgenein adult (5-6 weeks old) mouse tissues. Whole tissues were processed andstained for lacZ expression as previously described (Madsen et al. Circ.Res. 82:908-917, 1998). Results show that the human promoter hasactivity virtually identical to that of the rat SM-MHC promoter.

FIG. 10 shows histological evaluation of human MHC−5.1/13 in transgenicmice. Histological examination of specificity of expression of the humanMHC−5.1/13.5-LacZ transgene in adult (5-6 weeks old) mouse tissues.Tissues were processed and stained for lacZ expression as previouslydescribed (Madsen et al. Circ. Res. 82:908-917, 1998).

FIG. 11 shows nucleotide sequence comparison of the rat and human SM-MHCpromoter/enhancer sequence within the 5′ promoter region. As indicated,there is complete sequence homology between the rat and human genes inthe key regulatory regions identified thus far (e.g. 5′ CArG 1, 2 and 3;the G/C repressor, etc., as indicated). The identity of these elementsin the rabbit and mouse genes have been shown previously. See, Iadsen etal., 1997, J. Biol. Chem., 272:6332.

FIG. 12 shows gross examination of SM-MHC 4.2-Intron-lacZ expression invarious smooth muscle containing tissues. Transgenic mice (5-6 week-old)were perfusion fixed with a 2% formaldehyde/0.2% paraformaldehydesolution and various smooth muscle containing tissues were harvested andstained overnight at room temperature for β-galactosidase activity using5-bromo-chloro-3-indolyl-β-D galactopyranoside (X-Gal) as the substrate.

FIG. 13 shows histological analysis of rat SM-MHC 4.2-Intron-lacZexpression in various smooth muscle containing tissues. Transgenic mice(5-6 week-old) were perfusion fixed with a 2% formaldehyde/0.2%paraformaldehyde solution and various smooth muscle containing tissueswere harvested and stained overnight at room temperature forβ-galactosidase activity using5-bromo-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) as the substrate.After staining with X-Gal overnight, tissues were processed for paraffinembedding, sectioned at 6μm, and sections counterstained withhematoxylin/eosin.

FIG. 14 shows expression of SM-MHC 4.2-Intron-lacZ throughoutdevelopment. Embryos were harvested at various time points (10.5-16.5days p.c.), fixed with a 2% formaldehyde/0.2% paraformaldehyde solutionand stained overnight at room temperature for β-galactosidase activityusing 5-bromo-chloro-3-indolyl-β-D galactopyranoside (X-Gal) as thesubstrate. Embryos were then cleared in benzyl benzoate:benzyl alcohol(2:1). Panel A: 10.5 days p.c. Panel B: 12.5 days p.c. Panel C: 14.5days p.c. Panel D: 16.5 days p.c.

FIG. 15 shows expression of SM-MHC 4.2-Intron-lacZ at 19.5 days p.c.Embryos were harvested at 19.5 days p.c., fixed with a 2%formaldehyde/0.2% paraformaldehyde solution and stained overnight atroom temperature for β-galactosidase activity using5-bromo-chloro-3-indolyl -β-D-galactopyranoside (X-Gal) as thesubstrate. Embryos were then cleared in benzyl benzoate:benzyl alcohol(2:1). Panel A: Saggital section of 19.5 day embryo. Panel B: Closeup ofthoracic cavity. Panel C: Iliac artery and vein.

FIG. 16 shows expression of the SM-MHC 4.2-Intron-lacZ transgene in thecoronary circulation of the heart of an adult mouse. High levels ofSMC-specific expression are present in all major coronary arteries andarterioles.

FIG. 17 shows schematic representation of the rat SM-MHC 4.2-Intron-lacZclone and a comparable region of the human SM-MHC gene. As indicated,there is conservation of key regulatory elements including the CArGboxes, the GC repressor and an NF-1 site.

FIGS. 18A and 18B show mutants with deletions in the intronic CArGelement and their promoter activity. (A) A series of 3′-end deletionmutants of the SM-MHC LacZ sequence was generated and assayed forreporter activity in cultured rat SMCs. The β-galactosidase activity ofeach construct is expressed relative to the activity of the promoterlesspAUG LacZ. Error bars show standard error. (B) The nucleotide sequence(+1535 to +1703 from the transcription start site) of a portion of therat SM-MHC first intron (SEQ ID NO:16) was compared with thecorresponding human genomic sequence (GenBank U91323)(SEQ ID NO:17). Theintronic CArG element is boxed. Note that the human intronic CArG lacksa G-substitution within the central A/T-rich sequence and perfectlymatch the CArG consensus (CC(A/T)₆GG). Bold letters indicate the regionused in 3xICR TK LacZ construct. Nucleotides conserved with the ratsequence are indicated by dashes. Nucleotide additions are indicated bylower-case letters.

FIG. 19 show EMSA analysis of the CArG elements using tissue nuclearextracts. Radiolabeled 20-bp of double stranded oligonucleotidesencompassing CArG1, CArG2, intronic CArG, and c-fos SRE were incubatedwith either nuclear extracts prepared from tissues or SMCs orrecombinant serum response factor (SRF). The amount of nuclear extractswas determined to produce SRF shift bands of similar intensity: 4 μg ofaortic; 3 μg of bladder; 3 μg of stomach; 7 μg of heart; 3 μg of liver;and 5 μg of rat SMCs nuclear extracts. One μl of programmed lysate of invitro transcription/translation system (Promega) was used forrecombinant SRF.

FIG. 20 shows macroscopic examination of reporter gene expression inwild-type and mutant SM-MHC LacZ transgenic mice. Four-to 6-week-oldtransgenic mice were perfusion-fixed with a 2% formaldehyde/0.2%glutaraldehyde solution. Pictures show LacZ reporter expression invarious tissues from wild-type −4200/+11600 LacZ (A, E, I, M, Q), CArG1mutant (B, F, J, N, R), CArG2 mutant (C, G, K, O, S), and intronic CArGmutant mice (D, H, L, P, T). A-D, anterior view of the heart and aorta.E-H, the lung. I-L, the esophagus, stomach, and duodenum. M-P, a portionof small intestine. Q-T, the bladder. Tissues were cleared by benzylbenzoate/benzyl alcohol in A-H.

FIG. 21 shows large artery-specific silencing of the reporter gene inintronic CArG mutant mice. Abdominal organs removed en block showingreporter expression in the blood vessels and urinary tract in thewild-type (A) and intronic CArG mutant (B) transgenic mice. To betterillustrate transgene expression in large arteries, several smallerarteries and connective tissues were removed and the tissues cleared.The supramesenteric artery, which was stained positive, was removed fromthe intronic CArG mutant mouse tissues. A portion of the tissues isexpanded in the insert in panel B. Arrowheads indicate the position ofaorta that is not visible because of the lack of staining. Note that theblood vessels within the kidneys were not stained in either wild-typenor intronic CArG mutants. C, D, the thoracic aorta and branchingarteries of the wild-type (C) and the intronic CArG (D) mutanttransgenic mice. E, view of the large arteries in the cervicothoracicregion of the intronic CArG mutant transgenic mouse. F, the largearteries and their branches in the abdomen of the intronic CArG mutant.A portion of the arteries is expanded in the insert in F. G, H, I, J,histological examination of the abdominal aorta and inferior vena cavaof the wild-type (G, I) and the intronic CArG mutant (H, J) mice showingabrogation of reporter expression in SMCs of the aorta in the intronicCArG mutants. Note that expression in the vena cava was not changed bythe mutation. The boxed areas (G, H) are shown by a higher magnification(I, J). Ao indicates aorta; DA, ductus arteriosus; IVC, inferior venacava; SCA, subclavian artery.

FIG. 22 shows transgene expression in embryos. Embryos were harvested at19.5 dpc, skinned, and sectioned sagittally along the midline to permitdye penetration. The embryos were stained and cleared. The staining seenon the intestines in the negative and CArG2 mutant transgenic mice isdue to endogenous β-galactosidase activity and limited within theepithelial layer. Ao indicates aorta; Eso, esophagus; H, heart; St,stomach; Tr, trachea.

FIG. 23 shows supershift analysis of the intronic CArG-binding proteins.One μl of anti-SRF antibody was added to the binding reaction of anintronic CArG probe and nuclear extracts after 20 min of incubation onice and the reactions were further incubated for 10 min on ice. Additionof the antibody resulted in supershift of SRF-containing complexes (A,B). Complexes A and B formed with other CArG probes used in EMSAs inFIG. 23 were also supershifted (data not shown). Arrows indicatesupershifted complexes.

FIG. 24 shows chromatin immunoprecipitation analysis of SRF binding tothe endogenous CArG regions. PCR was carried out to detect theendogenous CArG regions in immunoprecipitated chromatin fragments. Lanes1, 4, 7, 10 show PCR amplification of control precipitation samples withno antibody. Lanes 2, 5, 8, and 11 shows amplification of 1:100 dilutionsamples of total input DNA for immunoprecipitation. Lanes 3, 6, 9, and12 show amplification of target sequences in immunoprecipitatedchromatin fragments with anti-SRF antibody.

FIG. 25 shows transgene expression of the intronic CArG region-minimalTK-LacZ. Various tissues of 4-week-old transgenic mice and embryos ofthe 3xICR-TK LacZ line (7240) were stained for β-galactosidase activity.A, B, anterior view of the heart and lung; C, the esophagus, stomach,and duodenum; D, a part of small intestine; E, a cross section of thebladder; F, anterior view of the kidneys, ureter, abdominal organs andgreat blood vessels; G, histological examination of the thoracic aorta;H, pulmonary artery and bronchus; I, cardiac muscle, coronary artery andintercostal muscle of the 3xICR-TK LacZ transgenic mice. Ao indicatesaorta; PA, pulmonary artery; SMA, superior mesenteric artery; IVC,inferior vena cava; H, heart; Br, bronchus; Eso, esophagus; Int,intestine; S, stomach; B1, bladder.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The invention provides novel isolated or recombinant polynucleotidescomprising cis-acting transcriptional control sequences of smooth muscle(SM) myosin heavy chain (SM-MHC) genes that confer smooth muscle cell(SMC) specific gene expression both in vitro (e.g., in cultured cells)and in vivo (e.g., in human or transgenic animals). The invention alsoprovides polynucleotides and expression vectors comprising SMC specifictranscription regulatory elements that are active in only certainsubtypes of SM cells. The polynucleotides of the invention include thosebased on or derived from genomic sequences of untranscribed, transcribedand intronic regions of SM-MHC genes, including the human SM-MHC(hSM-MHC) and rat SM-MHC (rSM-MHC) genes. Prior to the instantinvention, no genetic elements that are completely specific for SMC andwhich have been proven to confer smooth muscle specific gene expressionin vivo have been defined, isolated or identified. For example, thepreviously characterized SMC gene promoters, e.g., SM 22α and SM α-actinpromoters, all show activity in both SMC and non-SMC.

The invention also provides methods for using the SM-MHC promoters andother regulatory elements to control the expression of protein and RNAproducts in SMC. SM-MHC promoters and other regulatory elements have avariety of uses including, but not limited to, expressing heterologousgenes in SMC tissues, such as the contractile portion of the stomach,intestine and uterus, the walls of arteries, the ducts of secretoryglands and many other regions in which slow and sustained contractionsare needed. In addition, the targeted delivery is useful for developmentof animal models of human disease to assist in development of newtherapeutic targets or development of animal models for purpose ofscreening new drugs/therapies.

Another aspect of the invention relates to the use of SM-MHC promotersand other regulatory elements for genetic engineering as a means toinvestigate SMC physiology and pathophysiology. For example, a specificgene that is believed to be important for a specific disease within SMCcould be knocked out without the confounding influences of knocking outthat gene in other cell types and tissues. For example, an antisensepolynucleotide could be expressed under the control of an SM-MHCpromoter that would inhibit a target gene of interest, or an inhibitorcould be expressed that would specifically inhibit a particular protein.

The conventional (non-targeted) methods for gene knockout results inembryonic lethality, thus precluding the utility of studying involvementof these genes in control of SMC differentiation in diseases such asatherosclerosis, hypertension, and asthma. With the methods of thepresent invention, one could examine how selective (SMC-specific)knockout of an SMC gene of interest affects development of coronaryartery disease without the confounding limitations of conventionalknockouts with respect to deducing the primary site of action,activation of compensatory pathways, etc. Utilizing the SMC specificexpression vectors of the present invention, SMC specific gene knockoutcan be carried out using methods known in the art. The feasibility ofthese sorts of approaches has been shown in other non-SMC tissue types(see, e.g., Mayford et al., Science 274:1678, 1996). For example, theSM-MHC promoter/enhancers of the instant invention can be used incombination with the tetracycline-cre-recombinase based mouse systems toeffectuate targeted knockouts of various genes which are implicated inthe control of SMC differentiation within SMC tissues (Hautmann et al.,Circ. Res. 81:600,1997; Blank et al., Circ. Res. 76:742, 1995; Madsen etal, J. Biol. Chem. 272:6332,1997). Examples of such genes include geneswhich encode for serum response factor (SRF) (Kumar et al., J Biochem,118: 1285-92, 1995), the homeodomain protein MHox and the retinoic acidα-receptor.

A major biomedical application of the methods for SMC targeted genedelivery is to use the SM-MHC regulatory region to over-express a geneof interest within SMC. For example, an inhibitor of a pathologicprocess within an SMC tissue may be over-expressed in order to generatea high, local concentration of the factor that might be needed for atherapeutic effect. Since expression of the gene would be SMC-specific,undesired side effects on other tissues that often result whenconventional systemic administration of therapeutic agents are utilizedwould be avoided. For example, a gene for an SMC relaxant could beover-expressed within bronchiolar SMC as a therapy for asthma, or aninhibitor of SMC growth could be over-expressed to prevent developmentof atherosclerosis or post-angioplasty restinosis. Such applications ofthe present invention is exemplified in various embodiments disclosedherein. For example, FIG. 7 shows that a transgene under the control ofan SM-MHC promoter was specifically expressed at high levels within allcoronary arteries and arterioles within the heart of an adult mouse,demonstrating efficacy of the SM-MHC promoter/enhancer for gene therapyfor coronary artery disease.

The present invention also provides SM-MHC promoter/enhancers thatretain high level SMC specific expression in vivo and are selectivelyactive in subsets of SMC. Expression vectors containing such promotershave tremendous utility for targeting gene expression to specificsubtypes of smooth muscle in vivo. For example, these vectors can beemployed in targeting expression of a therapeutic gene to the specificsubtype of SMC desired (e.g. bronchiolar SMC for treatment of asthma orchronic bronchitis) thereby increasing the efficacy of the therapy andreducing potential side effects due to over-expression in undesiredtissues and cells. Efficacy of such applications of the presentinvention is demonstrated in, e.g., FIGS. 4-6 and 8, which showed thatsome SM-MHC promoters exhibit very high activity in subsets of SMCwithout loss of cell specificity.

Moreover, the SM specific promoter/enhancers sequences and expressionvectors of the present invention can also be employed in identificationand/or selection of smooth muscle cells derived from multi-potentialstem cell populations for purposes of tissue generation/regeneration forsurgery (e.g. for blood vessel, bladder, or gastrointestinal smoothmuscle tissue augmentation-reconstitution), and/or as a means ofdelivering a therapeutic gene to SMC tissues in vivo (as described inU.S. Provisional Application No. 60/277,202). The latter involves (i)introduction of a therapeutic gene into stem cells derived from asubject's bone marrow, adipose tissue or cryo-preserved umbilicalvessels; (ii) isolation and purification of SMC populations frommulti-potential stem cells using the SM-MHC promoter derivativesdescribed herein to drive expression of drug selectable markers such aspuromycin; and (iii) surgical introduction of the stem-cell-derived SMCinto the desired site of action in vivo.

A number of advantages are provided by the targeting methods of presentinvention. For example, SMC targeting will permit attainment of higherlocal concentrations of a therapeutic gene/agent at the desired site ofaction (i.e., SMC) than possible with systemic delivery methods, thusresulting in a greater therapeutic benefit and fewer possible sideeffects. In addition, SMC targeted gene therapy systems are much saferthan simple “restricted assess” gene delivery based methods that employconstitutively-active viral promoters, because the latter involvepotential accidental delivery of a therapeutic gene to an unintendedtissue or cell type may result in major undesirable side effects andpossible death. By contrast, an SMC specific promoter based targetingsystem is superior in that even if the therapeutic gene is delivered toan undesired cell type, it will not be expressed.

The following sections provide guidance for making and using thecompositions of the invention, and for carrying out the methods of theinvention.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY (1991). Although any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods and materialsare described. The following definitions are provided to assist thereader in the practice of the invention.

The terms “allele” or “allelic sequence” refer to an alternative form ofa polynucleotide sequence. Alleles result from mutations (i.e., changesin the polynucleotide sequence), and can produce differently regulatedmRNAs. Common mutational changes that give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, incombination with the others, or one or more times within a given gene,chromosome or other cellular polynucleotide.

The term “amplifying” incorporates its common usage and refers to theuse of any suitable amplification methodology for generating ordetecting recombinant or naturally expressed polynucleotide, asdescribed in detail, below. For example, the invention provides methodsand reagents (e.g., specific oligonucleotide PCR primer pairs) foramplifying (e.g., by PCR) naturally expressed or recombinantpolynucleotides of the invention (e.g., SM-MHC promoter/enhancersequences) in vivo or in vitro. An indication that two polynucleotidesare “substantially identical” can be obtained by amplifying one of thepolynucleotides with a pair of oligonucleotide primers or pool ofdegenerate primers (e.g., fragments of an SM-MHC promoter/enhancersequence) and then using the product as a probe under stringenthybridization conditions to isolate the second sequence (e.g., theSM-MHC promoter/enhancer sequence) from a genomic library or to identifythe second sequence in, e.g., a Northern or Southern blot.

A polynucleotide is “expressed” when a DNA copy of the polynucleotide istranscribed into RNA.

An “expression vector” is a polynucleotide construct, generatedrecombinantly or synthetically, with a series of specifiedpolynucleotide elements that permit transcription of a particularpolynucleotide in a host cell. The expression vector can be part of aplasmid, virus, or polynucleotide fragment. Typically, the expressionvector includes a polynucleotide to be transcribed operably linked to apromoter.

The term “heterologous” when used with reference to portions of apolynucleotide, indicates that the polynucleotide comprises two or moresubsequences which are not found in the same relationship to each otherin nature. For instance, the polynucleotide is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged ina manner not found in nature; e.g., an SM-MHC promoter sequence of theinvention operably linked to a polypeptide coding sequence that are nottranscribed from the SM-MHC genomic locus. For example, the inventionprovides recombinant constructs (expression cassettes, vectors, viruses,and the like) comprising various combinations of promoters of theinvention, or subsequences thereof, and heterologous coding sequences,many examples of which are described in detail below.

The terms “identical” or percent “identity,” in the context of two ormore polynucleotides or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides (or amino acid residues) that are the same,when compared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. This definitionalso refers to the complement of a sequence. For example, in alternativeembodiments, polynucleotides within the scope of the invention includethose with a nucleotide sequence identity that is at least about 60%, atleast about 75-80%, about 90%, and about 95% of the exemplary SM-MHCpromoter/enhancer sequence set forth in SEQ ID NO:16 or SEQ ID NO:17,and the intronic SM-MHC sequences capable of driving a reporter gene inSM cells, as described below. Two sequences with these levels ofidentity are “substantially identical.” Thus, if a sequence has therequisite sequence identity to an SM-MHC promoter/enhancer sequence orsubsequence of the invention, it also is an SM-MHC promoter/enhancersequence within the scope of the invention.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated or default program parameters. A “comparisonwindow” includes reference to a segment of any one of the number ofcontiguous positions selected from the group consisting of from 25 to600, usually about 50 to about 200, more usually about 100 to about 150in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well-knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection (see, e.g., Ausubel et al.,supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence (e.g., an SM-MHCpromoter/enhancer sequence of the invention as set forth by. e.g., SEQID NO:16 or SEQ ID NO:17) is compared to another sequence to determinethe percent sequence identity relationship (i.e., that the secondsequence is substantially identical and within the scope of theinvention) using the following parameters: default gap weight (3.00),default gap length weight (0.10), and weighted end gaps. PILEUP can beobtained from the GCG sequence analysis software package, e.g., version7.0 (Devereaux (1984) Nuc. Acids Res. 12:387-395).

Another example of algorithm that is suitable for determining percentsequence identity (i.e., substantial similarity or identity) is theBLAST algorithm, which is described in Altschul (1990) J. Mol. Biol.215:403-410. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always>0) and N (penalty score for mismatching residues,always<0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. In one embodiment, to determineif a polynucleotide sequence is within the scope of the invention, theBLASTN program (for nucleotide sequences) is used incorporating asdefaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4, anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as default parameters a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915).

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin (1993) Proc. Nat'l.Acad. Sci. USA 90:5873-5787). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, apolynucleotide is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test polynucleotide tothe reference polynucleotide is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

The term “isolated,” when referring to a molecule or composition, suchas, e.g., an SM-MHC promoter/enhancer sequence, means that the moleculeor composition is separated from at least one other compound, such as aprotein, DNA, RNA, or other contaminants with which it is associated invivo or in its naturally occurring state. Thus, a polynucleotidesequence is considered isolated when it has been isolated from any othercomponent with which it is naturally associated. An isolated compositioncan, however, also be substantially pure. An isolated composition can bein a homogeneous state. It can be in a dry or an aqueous solution.Purity and homogeneity can be determined, e.g., using analyticalchemistry techniques such as, e.g., polyacrylamide gel electrophoresis(PAGE), agarose gel electrophoresis or high pressure liquidchromatography (HPLC).

The term “modulate” refers to the suppression, enhancement or inductionof a function. For example, an agent or compound may modulate an SM-MHCpromoter/enhancer sequence by binding to a motif within thepromoter/enhancer, thereby enhancing or suppressing transcription of agene operably linked to the promoter/enhancer. Alternatively, modulationmay include inhibition of transcription of a gene where the an agent orcompound binds to the structural gene and blocks DNA dependent RNApolymerase from reading through the gene, thus inhibiting transcriptionof the gene. The structural gene may be a normal cellular gene or anoncogene, for example. Alternatively, modulation may include inhibitionof translation of a mRNA transcript.

The terms “nucleic acid” and “polynucleotide” are used interchangeably,and include oligonucleotides (i.e., short polynucleotides). They alsorefer to synthetic and/or non-naturally occurring polynucleotides (i.e.,comprising polynucleotide analogues or modified backbone residues orlinkages). The terms also refer to deoxyribonucleotide or ribonucleotideoligonucleotides in either single-or double-stranded form. The termsencompass polynucleotides containing known analogues of naturalnucleotides. The term also encompasses polynucleotide-like structureswith synthetic backbones. DNA backbone analogues provided by theinvention include phosphodiester, phosphorothioate, phosphorodithioate,methyl-phosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene (methylimino), 3′-N-carbamate, morpholinocarbamate, and peptide polynucleotides (PNAs); see Oligonucleotides andAnalogues, a Practical Approach, edited by F. Eckstein, IRL Press atOxford University Press (1991); Antisense Strategies, Annals of the NewYork Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Researchand Applications (1993, CRC Press). PNAs contain non-ionic backbones,such as N-(2-aminoethyl)glycine units. Phosphorothioate linkages aredescribed in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.Pharmacol. 144:189-197. Other synthetic backbones encompassed by theterm include methyl-phosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (Strauss-Soukup (1997)Biochemistry 36:8692-8698), and benzyl-phosphonate linkages (Samstag(1996) Antisense Nucleic Acid Drug Dev 6:153-156).

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, it refers tothe functional relationship of a transcriptional regulatory sequence toa transcribed sequence. For example, an SM-MHC promoter/enhancersequence of the invention, including any combination of cis-actingtranscriptional control elements, is operably linked to a codingsequence if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance. A polylinker provides aconvenient location for inserting coding sequences so the genes areoperably linked to the SM-MHC promoter. Polylinkers are polynucleotidesequences that comprise a series of three or more closely spacedrestriction endonuclease recognition sequences.

The promoter region of a gene includes the regulatory elements thattypically lie 5′ to a structural gene. If a gene is to be activated,proteins known as transcription factors attach to the promoter region ofthe gene. This assembly resembles an “on switch” by enabling an enzymeto transcribe a second genetic segment from DNA into RNA. In most casesthe resulting RINA molecule serves as a template for synthesis of aspecific protein; sometimes RNA itself is the final product. Thepromoter region may be a normal cellular promoter or an oncopromoter.

The term “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. “Recombinant means”also encompass the ligation of polynucleotides having coding or promotersequences from different sources into an expression cassette or vectorfor expression of, e.g., a fusion protein; or, inducible, constitutiveexpression of a protein (i.e., an SM-MHC promoter/enhancer of theinvention operably linked to a heterologous nucleotide, such as apolypeptide coding sequence).

The “sequence” of a gene (unless specifically stated otherwise) orpolynucleotide refers to the order of nucleotides in the polynucleotide,including either or both strands of a double-stranded DNA molecule,e.g., the sequence of both the coding strand and its complement, or of asingle-stranded polynucleotide molecule. For example, the promoter ofthe invention comprises untranscribed, untranslated, and intronic SM-MHCsequences, e.g., as set forth in the exemplary SEQ ID NO:16 and SEQ IDNO:17.

Unless otherwise specified, the term “SM-MHC” broadly refers to smoothmuscle myosin heavy chain, as well as the corresponding polynucleotideand polypeptide sequences. See White et al., J. Biol. Chem.27115008-15017, 1996.

Unless otherwise specified, the terms “SM-MHC promoter,” “SM-MHCpromoter/enhancer” and “SM-MHC promoter/enhancer sequence” are usedinterchangeably and refer to a polynucleotide which comprises SM-MHCgenomic sequence and activates transcription of a linked polynucleotidein smooth muscle cells in vitro and in vivo. Unless otherwise noted, theSM-MHC promoter/enhancers of the present invention do not include apolynucleotide which can drive DNA expression in cultured SMCs, but notin an animal having a smooth muscles (e.g., transgenic mice). The SM-MHCpromoter/enhancer sequences can include all cis-acting SM-MHCtranscriptional control elements and regulatory sequences, including(without limitation) those that regulate and modulate timing and ratesof transcription. Thus, the SM-MHC promoter/enhancer sequences of theinvention can include cis-acting elements such as, e.g., promoters,enhancers, transcription terminators, origins of replication,chromosomal integration sequences, introns, exons, and 5′ and 3′untranslated regions, with which proteins or other biomolecules interactto carry out and regulate transcription of the SM-MHC transcript.

The term “smooth muscle-specific expression” or “smooth muscle-specifictranscription” means that a polynucleotide is transcribed at a greaterrate in smooth muscle cells than in non-smooth muscle cells. ExemplarySM cells include cells which form the contractile portion of thestomach, intestine and uterus, the walls of arteries, the ducts ofsecretory glands and many other regions in which slow and sustainedcontractions are needed. In general, an SM specific promoter and/orenhancer will generally activate transcription of a linkedpolynucleotide at least 3-fold more efficiently in SM cells than innon-SM cells. In certain embodiments, transcription is at least 3-fold,5-fold, 10-fold, 25-fold or 100-fold more efficient in SM cells than innon-SM cells. Unless otherwise specified, SM-MHC promoter/enhancers ofthe present invention do not have detectable activity in non-SM cellswhen examined using a reporter gene (e.g., lacZ) as described in theExamples. SM-specific transcription may result from an increasedfrequency of transcriptional initiation, an increased rate oftranscriptional elongation, a decreased frequency of transcriptionaltermination, or a combination thereof.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule to a particularnucleotide sequence under moderately or highly stringent hybridizationconditions when that sequence is present in a complex mixture (e.g.,total cellular or library DNA or RNA), wherein the particular nucleotidesequence is detected at least twice background, preferably 10 timesbackground. In one embodiment, a polynucleotide can be determined to bewithin the scope of the invention (e.g., is substantially identical toan SM-MHC promoter/enhancer of the invention, as exemplified by SEQ IDNO:16 or SEQ ID NO:17, or, by an intronic promoter sequence, asdescribed below) by its ability to hybridize under stringent conditionsto another polynucleotide (such as the exemplary sequences describedherein).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will primarily hybridize to its target subsequence,typically in a complex mixture of polynucleotide, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances, e.g., depending on the length ofthe probe. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of polynucleotidesis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to about 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than about 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal (e.g., identification of a polynucleotide of theinvention) is about 5-10 times background hybridization. “Stringent”hybridization conditions that are used to identify substantiallyidentical polynucleotides within the scope of the invention includehybridization in a buffer comprising 50% formamide, 5×SSC, and 1% SDS at42° C., or hybridization in a buffer comprising 5×SSC and 1% SDS at 65°C., both with a wash of 0.2×SSC and 0.1% SDS at 65° C., for long probes.For short probes, stringent hybridization conditions includehybridization in a buffer comprising 50% formamide, 5×SSC and 1% SDS atroom temperature or hybridization in a buffer comprising 5×SSC and 1%SDS at 37° C.-42° C., both with a wash of 0.2×SSC and 0.1% SDS at 37°C.-42° C. However, as is apparent to one of ordinary skill in the art,hybridization conditions can be modified depending on sequencecomposition. Exemplary “moderately stringent hybridization conditions”include a hybridization in a buffer of 40% formamide, 1 M NaCl, and 1%SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization isat least twice background. Those of ordinary skill will readilyrecognize that alternative hybridization and wash conditions can beutilized to provide conditions of similar stringency.

“Transcription initiation elements” refer to sequences in a promoterthat specify the start site of RNA polymerase II. Transcriptioninitiation elements may include TATA boxes, which direct initiation oftranscription 25-35 bases downstream, or initiator elements, which aresequences located near the transcription start site itself. Eukaryoticpromoters generally comprise transcription initiation elements andeither promoter-proximal elements, distant enhancer elements, or both.SM-MHC transcription initiation elements may include the TATA box ortranscription initiation sites described herein, or both. Heterologoustranscription initiation elements may be obtained from any eukaryoticpromoter, although mammalian and viral promoters are preferred sourcesof heterologous initiation elements.

The term “transcribable sequence” refers to any sequence which, whenoperably linked to a cis-acting transcriptional control element, e.g., apromoter, such as the SM-MHC promoter/enhancers of the invention, andwhen placed in the appropriate conditions, is capable of beingtranscribed to generate RNA, e.g., messenger RNA (mRNA).

III. Polynucleotides Comprising Smooth Muscle SpecificPromoter/Enhancers

The present invention provides polynucleotide sequences which confer toan operably linked polynucleotide cell-specific expression within SMcells in vivo. These polynucleotide sequences, termed SM-MHCpromoter/enhancers, are derived from the smooth muscle myosin heavychain (SM-MHC) gene. Some of the SM-MHC promoter/enhancers are obtainedfrom the human SM-MHC sequence (e.g., SEQ ID NO:17). SEQ ID NO:17contains residues −5086 to +13,518 of the human SM-MHC gene sequence.Nucleotide 1 in SEQ ID NO:17 corresponds to position −5086 relative tothe transcription start site (+1 position) which in turn corresponds toposition 143,590 in the undefined BAC sequence contained in the publicdatabase (GenBank Accession No. U91323). Some of the SM-MHCpromoter/enhancers are derived from rat SM-MHC sequence (e.g., SEQ IDNO:16). Nucleotide 1 of SEQ ID NO:16 corresponds to position −4,216 bprelative to the SM-MHC transcription start site.

The present invention also provides SM specific promoter/enhancers whichare active only in certain subsets of smooth muscle tissues. Some ofthese SM-MHC promoter/enhancers comprise a polynucleotide sequence whichconsists essentially of the region of nucleotides 5663 to 5889 of SEQ IDNO:16 (the +1447 to +1673 intronic region). Other comprise a sequence ofSEQ ID NO:16 except that the CArG2 or the intronic CArG motif has beenmutated. Some of the promoter/enhancers comprise a polynucleotidesequence which consists essentially of the regions of nucleotides 1 to6,700 (the −4.2 to +2.5 region) and nucleotides 9,500 to 15,800 (the+5.3 to +11.6 region) of SEQ ID NO:16. Some of the subset-specificSM-MHC promoter/enhancers comprise a polynucleotide sequence whichconsists essentially of the regions of nucleotides 1 to 9,500 (the −4.2to +5.3 region) and nucleotides 11,700 to 13,700 (the +7.5 to +9.5region) of SEQ ID NO:16.

In alternative embodiments, the SM-MHC promoter/enhancer sequencescomprise sequences substantially identical to an exemplary SM-MHCpromoter/enhancer sequence as discussed above. Thus, SM-MHCpromoters/enhancers of the instant invention include homologous SMCpromoter/enhancer elements which have similar functional activity. Thisincludes SMC promoters/enhancers which direct SMC-specific expression invivo and either hybridize to the above-described SM-MHCpromoter/enhancers under highly stringent conditions, or that hybridizeto the complement of the above-described promoter/enhancers undermoderately stringent conditions.

SM-MHC promoter/enhancer sequences can range from 100 to 20,000nucleotides in length, although in particular embodiments functionalSM-MHC promoter/enhancer polynucleotides may be at least or no more thanabout 300, 500, 1,000, 2,500, 5,000, 10,000, or 15,000 nucleotides inlength. SM-MHC promoter/enhancer polynucleotides of the presentinvention are generally at least 70% homologous to SEQ ID NO:16 or SEQID NO:17 over a stretch of 150 nucleotides or more. In some embodiments,SM-MHC promoter/enhancer polynucleotides are at least 75%, 80%, 85%,90%, 92%, 95%, or 100% homologous to SEQ ID NO:16 or SEQ ID NO:17 over astretch of 300, 500, 1,000, 2,500, 5,000, 10,000 or 15,000 nucleotides.

As detailed in the Examples, some of the SM-MHC promoter/enhancersequences comprise non-transcribed SM-MHC genomic sequence as well aseither SM-MHC introns or exons, or both. In some embodiments, SM-MHCpromoter/enhancer polynucleotides include the SM-MHC TATA box andtranscription initiation sites (collectively referred to as SM-MHCtranscription initiation elements). In embodiments where the SM-MHCtranscription initiation elements are the only functional initiationelements of the promoter, the natural orientation of the SM-MHC TATA boxor transcription initiation sites, relative to the direction oftranscription, should be preserved. In other embodiments, SM-MHCpromoter/enhancer polynucleotides are connected to heterologous TATAboxes and/or transcription initiation sites. When linked to heterologousTATA boxes or transcription initiation sites, SM-MHC promoter/enhancerpolynucleotides act as enhancer elements and may be inserted in eitherorientation relative to the direction of transcription. Thus, the term“SM-MHC promoter/enhancer” encompasses polynucleotides comprising thetranscription initiation elements of the SM-MHC gene, as well ascis-linked enhancer sequences that yield smooth muscle-specificexpression when linked to the transcription initiation elements of aheterologous gene.

A. Isolation of SM Specific Promoter/Enhancer Sequences

1. Isolation of SM-MHC Promoter Sequences

The SM-MHC promoter/enhancer sequences of the invention andpolynucleotides used to practice this invention, whether RNA, cDNA,genomic DNA, or hybrids thereof, may be isolated from a variety ofsources, genetically engineered, amplified, and/or expressedrecombinantly. Any recombinant expression system can be used, including,e.g., bacterial, yeast, insect or mammalian systems. Alternatively,these polynucleotides can be chemically synthesized in vitro.

In some embodiments, SM-MHC promoter sequences are isolated fromlibraries of genomic DNA. Some genomic libraries are commerciallyavailable. For example, rat genomic phage library can be obtained fromStratagene Corp. Genomic DNA libraries are also available from variousother commercial suppliers (e.g., Incyte Genomics, Palo Alto, Calif.;Clontech, Palo Alto, Calif.). Alternatively, genomic libraries can alsobe constructed, e.g., as described in Ausubel et al., supra. For agenomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

In some embodiments, the SM-MHC promoter/enhancer sequences are obtainedfrom genomic clones containing 5′ flanking region and the intronicregions of the SM-MHC gene. Standard methods that may used in suchscreening include, for example, the method set forth in Benton & Davis,1977, Science 196:180 for bacteriophage libraries; and Grunstein &Hogness, 1975, Proc. Nat. Acad. Sci. U.S.A. 72:3961-3965 for plasmidlibraries.

SM-MHC promoter polymorphic variants, orthologs, and alleles that aresubstantially identical to SM-MHC promoter sequences can be isolatedusing SM-MHC promoter/enhancer polynucleotide probes andoligonucleotides under stringent hybridization conditions, by screeninglibraries from the appropriate organism.

Techniques for the manipulation of polynucleotides, such as, e.g.,subcloning into expression vectors, labeling probes, sequencing, andhybridization are well described in the scientific and patentliterature, see e.g., ed., Molecular Cloning: A Laboratory Manual (2ndEd.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) (“Sambrook”);Current Protocols In Molecular Biology, Ausubel, ed. John Wiley & Sons,Inc., New York (1997) (“Ausubel”); Laboratory Techniques In BiochemistryAnd Molecular Biology: Hybridization With Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993)(“Tijssen”). Nucleic acids can be analyzed and quantified by any of anumber of general means well known to those of skill in the art. Theseinclude, e.g., analytical biochemical methods such as NMR,spectrophotometry, radiography, electrophoresis, capillaryelectrophoresis, high pressure liquid chromatography (HPLC), thin layerchromatography (TLC), and hyperdiffusion chromatography, variousimmunological methods, such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, Southern analysis, Northern analysis,dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR,quantitative PCR, other polynucleotide or target or signal amplificationmethods, radiolabeling, scintillation counting, and affinitychromatography.

Oligonucleotides that are not commercially or publicly available can bechemically synthesized according to the solid phase phosphoramiditetriester method first described by Beaucage & Caruthers, TetrahedronLetts. 22:1859-1862 (1981), using an automated synthesizer, as describedin Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984).Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255:137-149 (1983).

Synthetic oligonucleotides can be also used to construct recombinantSM-MHC promoter sequences for use as probes or for generation of smoothmuscle-specific promoters. This method is performed using a series ofoverlapping oligonucleotides usually 40-120 bp in length, representingboth the sense and non-sense (antisense) strands of the gene. These DNAfragments are then annealed, ligated and cloned. Alternatively,amplification techniques can be used with precise primers to amplify aspecific subsequence of an SM-MHC promoter sequence.

SM-MHC promoter sequences are typically cloned into intermediate vectorsbefore transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryotic vectors, e.g., plasmids, or shuttle vectors.

2. Modification of SM-MHC Promoter Sequences

Once smooth muscle-specific transcriptional activity has beendemonstrated in an SM-MHC promoter/enhancer sequence, deletions,mutations, rearrangements, and other sequence modifications may beconstructed and analyzed for smooth muscle-specific transcription. Suchderivatives of SM-MHC promoter sequences are useful to generate morecompact promoters, to decrease background expression in non-smoothmuscle cells, to eliminate repressive sequences, or to identify novelsmooth muscle-specific transcriptional regulatory proteins.

SM-MHC promoter subfragments and derivatives may be constructed byconventional recombinant DNA methods known in the art. One such methodis to generate a series of deletion derivatives within the promotersequence (see, e.g., FIG. 18A and Example 2). By comparing thetranscriptional activity of a deletion series, the elements thatcontribute to or detract from smooth muscle-specific transcription maybe localized. Based on such analyses, improved derivatives of SM-MHCpromoter sequences may be designed. SM-MHC promoter elements may becombined with smooth muscle-specific or ubiquitous regulatory elementsfrom heterologous promoters to increase the specificity or activity ofan SM-MHC promoter sequence.

The modified SM-MHC promoter/enhancer sequences can contain deletion inone or more of the cis-acting elements. Cis-acting regulatory elementswithin a promoter/enhancer may be identified using methods such as DNaseor chemical footprinting (e.g. Meier et al., 1991, Plant Cell 3:309-315)or gel retardation (e.g., Weissenbom & Larson, 1992, J. Biol. Chem.267-6122-6131; Beato, 1989, Cell 56:335-344; Johnson et al., 1989, Ann.Rev. Biochem. 58:799-839). Additionally, resectioning experiments alsomay be employed to define the location of the cis-regulatory elements.For example, a promoter/enhancer containing fragment may be resectedfrom either the 5′ or 3′ end using restriction enzyme or exonucleasedigests.

In addition, specific base pairs can be modified to alter, increase ordecrease the binding affinity to trans-acting transcriptional regulatoryfactors, thus modifying the relative level of transcriptional activationor repression. Modifications can also change secondary structures ofspecific subsequences, such as those associated with many cis-actingtranscriptional elements. Site-specific mutations can be introduced intopolynucleotides by a variety of conventional techniques, well describedin the scientific and patent literature. Illustrative examples include,e.g., site-directed mutagenesis by overlap extension polymerase chainreaction (OE-PCR), as described in Urban (1997) Nucleic Acids Res.25:2227-2228; Ke (1997) Nucleic Acids Res 25:3371-3372, andChattopadhyay (1997) Biotechniques 22:1054-1056. Modified SM-MHCpromoter/enhancer sequences of the invention can be further produced bychemical modification methods, see, e.g., Belousov (1997) Nucleic AcidsRes. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380;Blommers (1994) Biochemistry 33:7886-7896.

B. Activity of SM-MHC Promoter/Enhancers

The present invention provides smooth muscle-specific SM-MHC promotersand enhancers. Accordingly, methods for assaying the smoothmuscle-specific transcription induced by SM-MHC promoter sequences areprovided herein.

Promoter activity of an SM-MHC promoter sequence is generally assayed byoperably linking the SM-MHC promoter sequence to a reporter gene (e.g.,a lacZ gene) in a test construct (see, e.g., Example 1, infra). Wheninserted into the appropriate host cell (e.g., cultured rat SM cells),the SM-MHC promoter sequence induces transcription of the reporter geneby host RNA polymerases. Reporter genes typically encode proteins (e.g.,β-galactosidase) with an easily assayed enzymatic activity that isnaturally absent from the host cell. Alternatively, endogenous activityof the reporter protein can be measured with a control construct whichdoes not express the reporter gene, and substracted from the activitymeasured for the test construct. In addition to β-galactosidase, otherreporter proteins that can be applied in the present invention includechloramphenicol acetyltransferase (CAT), firefly or Renilla luciferase,β-galactosidase, beta-glucuronidase, alkaline phosphatase, and greenfluorescent protein (GFP). In some embodiments, SM-MHC promoterfragments can be inserted into a polylinker sequence and tested foractivity of the reporter protein in the appropriate host cell (see,e.g., U.S. Pat. No. 5,670,356).

Transcription driven by SM-MHC promoter sequences can also be detectedby directly measuring the amount of RNA transcribed from the reportergene. In these embodiments, the reporter gene can be any transcribablepolynucleotide of known sequence that is not otherwise expressed by thehost cell. RNA expressed from SM-MHC promoter constructs may be analyzedby techniques known in the art, e.g., reverse transcription andamplification of MRNA, isolation of total RNA or poly A⁺ RNA, northernblotting, dot blotting, in situ hybridization, RNase protection, primerextension, high density polynucleotide array technology and the like.

In addition to reporter genes, vectors for assaying SM-MHC promotersequence activity also comprise elements necessary for propagation ormaintenance in the host cell, and elements such as polyadenylationsequences and transcriptional terminators to increase expression ofreporter genes or prevent cryptic transcriptional initiation elsewherein the vector. Assay vectors may also comprise other transcriptionregulatory (e.g., transcription initiation) sequences, depending onwhether the SM-MHC transcription initiation elements are included in theSM-MHC promoter sequence being assayed.

1. Assaying Activity of SM-MHC Promoter/Enhancers SM Cells

The ability of a promoter sequence to activate transcription can beassessed relative to a control construct which harbors a referencepromoter. In some embodiments, the specificity of an SM-MHC promotersequence to activate transcription is assessed by comparing theexpression of a reporter gene operably linked to an SM-MHC promotersequence with the expression of the identical reporter gene operablylinked to a reference promoter. For example, the activity of a reportergene driven by an SM-MHC promoter sequence can be compared to theactivity of a reporter gene driven by a characterized promoter (e.g.,the SV40 promoter/enhancer, Promega, Madison, Wis.).

SM-MHC promoter sequences of the present invention are smoothmuscle-specific, activating transcription to a greater extent in smoothmuscle cells than in non-smooth muscle cells. Accordingly, smooth musclespecificity of an SM-MHC promoter sequence may be assessed by assayingits promoter or enhancer activity in a smooth muscle cell and anon-smooth muscle cell. In some embodiments, the assay for smoothmuscle-specific promoter activity generally requires simultaneouscomparison of reporter gene expression in four contexts: the testpromoter in a smooth muscle cell, a reference promoter (e.g., lackingSM-MHC sequences) in the smooth muscle cell, the test promoter in anon-smooth muscle cell, and the reference promoter in a non-smoothmuscle cell. Once the promoter activity of the SM-MHC polynucleotide ineach cell type is determined by comparing the test promoter and thereference promoter, the smooth muscle specificity of the SM-MHCpolynucleotide is calculated by comparing the activity of the testpromoter in the smooth muscle cell with its activity in a non-smoothmuscle cell.

One system for assessing SM-MHC promoter activity is transient or stabletransfection into cultured cell lines. Assay vectors bearing SM-MHCpromoter sequences operably linked to reporter genes can be transfectedinto any mammalian cell line for assays of promoter activity. Suitablemethods of cell culture, transfection, and reporter gene assay aredescribed in, e.g., Ausubel et al., supra; or Transfection Guide,Promega Corporation, Madison, Wis. (1998). SM-MHC promoter sequences maybe assayed for smooth muscle-specific transcription activity bytransfecting the assay vectors in parallel into smooth muscle cell linesand non-smooth muscle cell lines. In some embodiments, a control vectorcomprising a second reporter gene driven by a known promoter (e.g.,Renilla luciferase driven by the SV40 early promoter/enhancer; pRL-SV40,Promega, Madison, Wis.) is co-transfected along with the assay vector tocontrol for variations in transfection efficiency or reporter genetranslation among the smooth muscle and non-smooth muscle cell lines.

2. Assaying In Vivo Activity of the SM-MHC Promoter/Enhancers

As disclosed above, the activity of specificity of the SM-MHCpromoter/enhancers of the present invention can be assayed in eukaryoticin vitro transcription systems (e.g., cultured rat SM cells). Theiractivity can also be examined in transgenic animals (e.g., transgenicmice). Further, it is known that some promoter or enhancers withspecificity in cultured SM cells do not have activity in vivo, e.g., intransgenic mice. Thus, to determine in vivo specificity, the SM-MHCpromoter/enhancers are also assayed for their activity in transgenicanimals.

Transgenic animals (e.g., transgenic mice) expressing SM-MHCpromoter/enhancer can be generated accordingly to methods well known inthe art (see, e.g., Example 1). For example, techniques routinely usedto create and screen for transgenic animals have been described in,e.g., see Bijvoet (1998) Hum. Mol. Genet. 7:53-62; Moreadith (1997) J.Mol. Med. 75:208-216; Tojo (1995) Cytotechnology 19:161-165; Mudgett(1995) Methods Mol. Biol. 48:167-184; Longo (1997) Transgenic Res.6:321-328; U.S. Pat. No. 5,616,491 (Mak, et al.); U.S. Pat Nos.5,464,764; 5,631,153; 5,487,992; 5,627,059; 5,272,071; and, WO 91/09955,WO 93/09222, WO 96/29411, WO 95/31560, and WO 91/12650.

Transgenic animals with integrated SM-MHC promoter sequences can be usedto assay for SM specific transcription. In some embodiments, an SM-MHCpromoter sequence, linked either to a reporter gene or to native SM-MHCcoding sequence, is injected into the embryo of a developing animal(typically a mouse) to generate a transgenic animal. Once integration ofthe transgene has been verified, smooth muscle and non-smooth muscletissues of the animal are then assayed for expression of the transgenewith conventional RNA or protein detection methods known in the art anddescribed herein. Typically, a rat or a human SM-MHC promoter sequenceis employed, in which case RNA expressed from the transgene may bedistinguished from RNA expressed from the endogenous mouse SM-MHC locusby employing appropriate polynucleotide probes that are specific for therat or human SM-MHC sequence. Alternatively, where the SM-MHC promotersequence is linked to a reporter gene, tissues of the transgenic animalmay be assayed either for reporter gene RNA or for the enzymaticactivity of the reporter protein (see, e.g., Examples 1, 2 and 4).

C. Exemplary SM-MHC Promoter/Enhancers

The SM specific promoter disclosed herein can be obtained as describedin the Examples, e.g., cloned from genomic DNA libraries or isolatedusing amplification techniques with oligonucleotide primers. Anexemplary SM specific promoter is the rat −4.2 to +11.6 regionpromoter/enhancer of rat SM-MHC (SEQ ID NO:16) (see also, Madsen et al.,Circ. Res. 82:908-917, 1998). The corresponding human SM-MHCpromoter/enhancer sequence has also been identified (the −5,086 to+13,518 fragment; SEQ ID NO:17). Other than the 5′-flanking sequence(e.g., residues 1-4216 of SEQ ID NO:16) and the short first exon (e.g.,the 88 bp exon in SEQ ID NO:16), these SM-MHC promoter/enhancersequences also contain portion of the first intron of the SM-MHC gene.

Some of the SM-MHC promoter/enhancers confer specificity in all SMcells. For example, the −4.2 to +11.6 kb fragment of the SM-MHCpromoter/enhancer (corresponding to residues 1-16011 of SEQ ID NO:16)exhibits high level activity in virtually all SMC subtypes (FIGS. 1-3and 12). Transgene expression under control of this promoter wasobserved in both arterial and venous smooth muscle, airway smooth muscleof the trachea and bronchi and in the smooth muscle layers of allabdominal organs, including the stomach, intestine, ureters and bladder.In addition, the transgene was expressed at high levels throughout thecoronary circulation (see, FIG. 7). During development, transgeneexpression was first detected in airway SMC at embryonic day 12.5 and invascular and visceral SMC tissues by embryonic day 14.5.

Human SM-MHC promoter/enhancer with in vivo SM specificity was alsoidentified by the present inventors. As disclosed in Examples 2-4 andFIGS. 9-10), the region from −5086 to +13518 of the human SM-MHC gene(SEQ ID NO:17) was found highly active in multiple SMC tissues, butexhibited absolutely no expression in non-SMC tissues. This includedexpression in SMC within multiple small and large vessels including theaorta, coronary arteries, illiac, celiac, mesenteries, etc. Thispromoter was also robustly expressed in SMC within the stomach,intestine, bladder, ureter, and airways. As illustrated in FIGS. 11, 17and 18, there is complete sequence homology between the rat and humangenes in the key regulatory regions identified thus far (e.g. 5′ CArG 1,2 and 3; the G/C repressor, etc., as indicated). The identity of theseelements in the rabbit and mouse genes have been shown previously (e.g.,Iadsen et al., 1997, J. Biol. Chem., 272:6332).

Other than conferring specificity in all SM tissues, some SM-MHCpromoter/enhancers confer SM specificity only in selective subtypes ofSM tissues (i.e. vascular versus gastrointestinal SMC, large versussmall arteries, pulmonary versus gastrointestinal SMC, etc.) (see,Examples 2-4, FIGS. 4-8). Various derivative SM-MHC promoter/enhancersobtained from the −4.2 to +11.6 kb rat SM-MHC promoter/enhancer regionwere found to be active in one or more, but not all subtypes of SMtissues. For example, some of such SM-MHC promoter/enhancers includethose that comprise essentially the sequence of nucleotides 5663 to 5889of SEQ ID NO:16 (corresponding to the intronic region of +1447 to+1673). This fragment contains three repeats of the intronic region ofSM-MHC, and when coupled to a minimal thymidine kinase (TK) promoter,confers high level expression in multiple SMC tissues including theaorta, coronary arteries, and pulmonary artery (See FIG. 7).

Some of the subtype-specific SM-MHC promoter/enhancers have an excisionof the region from +5.3 to +11.6 kb. These promoters do not confer SMexpression in vascular SMC but retain the activity in gastrointestinal,and bladder SMC. Some of these subtype-specific SM-MHCpromoter/enhancers consists essentially the regions −4.2 to +5.3 and+7.5 to +9.5 (corresponding to residues 1 to 9,500 and nucleotides11,700 to 13,700 of SEQ ID NO:16), or −4.2 to +2.5 and +5.3-11.6(corresponding to residues 1 to 6,700 and nucleotides 9,500 to 15,800 ofSEQ ID NO:16). They exhibit very high activity in both pulmonaryvascular and airway SMC (see FIGS. 4-6).

In still some other SM-MHC promoter/enhancers, subtype-specificity isconferred by the deletion of certain conserved motifs (e.g., theintronic CArG motif or the CArG2 motif). For example, some of thepromoter/enhancers have a mutation in the conserved intronic CArGelement (i.e., residues 5815-5824 of SEQ ID NO:16). An exemplary mutanthas the intronic CArG sequence changed from CCTTGTATGG (SEQ ID NO:5) toAGGCCTATGG (SEQ ID NO:6). The mutation abolishes promoter activity inthe aorta, coronary arteries, and the carotid artery, without affectingexpression in other SMC tissues including pulmonary vascular or airwaySMC (see, e.g., FIG. 8). Some other SM-MHC promoters have a mutation inthe CArG2 motif (i.e., residues 3105-3114 of SEQ ID NO:16). An exemplarypromoter having such a mutation has the CArG2 sequence changed fromTTCCTTTTATGG (SEQ ID NO:1) to GGATCCTATGG (SEQ ID NO:2).

D. Expression Vectors and Transgenic Animals

The invention provides expression vectors for targeted gene delivery andexpression in SM cells. The expression vectors comprise an SM-MHCpromoter/enhancer sequence operably linked to a heterologous gene (in apreferred embodiment, a structural gene). The heterologous codingsequence operably linked to an SM-MHC promoter/enhancer of the inventioncan be a marker or reporter gene (e.g., alkaline phosphatase, SEAP;β-galactosidase), a modified SM-MHC structural gene or an SM-MHCantisense sequence, a therapeutic gene. Other than the promoter and aheterologous gene, the vectors can also comprise other elements, e.g.,origins of replication. These constructs are useful for SM-MHCpromoter-based assays, for example, to identify biological modulators ofSM-MHC promoter/enhancer activity.

Some of the SMC specific expression vectors of the present inventioncomprise an SM-MHC promoter sequence described above. Some of theexpression vectors contain the polynucleotide sequence of SEQ ID NO:16or 17. Some expression vector contain an SM-MHC promoter/enhancer whichconsists essentially of one of the following sequences

1) the intronic region from +1447 to +1673 (residues 5663 to 5889) ofSEQ ID NO:16;

2) the region of −4.2 to +11.6 of SEQ ID NO:16, wherein CArG2 orintronic CArG have been mutated;

3) the regions of −4.2 to +2.5 and +5.3 to +11.6 (residues 1 to 6,700and nucleotides 9,500 to 15,800)of SEQ ID NO:16; and

4) the regions of −4.2 to +5.3 and +7.5 to +9.5 (residues 1 to 9,500 andnucleotides 11,700 to 13,700 of SEQ ID NO:16) of SEQ ID NO:16.

As discussed in more detail below, these expression vectors are usefulfor targeting gene expression specifically to smooth muscle or subtypesof smooth muscle, development of animal models of human disease for drugscreening, or elucidation of pathogenic mechanisms and identification ofnew therapeutic targets.

Employing aforementioned expression vectors, the present inventionprovides host cells and transgenic animals which have incorporated aheterologous polynucleotide in SM cells. Such host cells or transgenicanimals (e.g., transgenic mice) of the present invention can be producedas described above and in the Examples. The transgenic cells or animalsof the present invention can be used in various applications, e.g.,development of animal models for purpose of screening newdrugs/therapies. For example, if a specific gene is known to be involvedin an SMC-based disease, the gene can be operably linked to an SM-MHCpromoter/enhancer of the instant invention to produce an animal model ofthe disease.

In addition, transgenic cells or animals of the present invention canalso comprise an SM-MHC promoter/enhancer operably linked to a genewhich expresses a protein which can inhibit (a) other proteins or (b)transcription of other genes that further the diseased state beingexamined within the animal model. Alternatively, the SM-MHCpromoter/enhancer can be operably linked to an antisense gene, whichcould specifically inhibit expression of a gene which may be involved inthe diseased state. Using such animal models, one of skill in the artcould test conventional drug therapies, identify key genes involved inthe development of these diseases and/or develop a novel way of curingthe disease.

IV. Targeted Gene Delivery and Expression

The present invention provides methods for targeted delivery oftherapeutic agents to SM cells in a subject (human or non-humananimals). The therapeutic agents include polynucleotides that arespecifically expressed in vivo under the control of the SM-MHCpromoter/enhancers. Virtually any gene can be specifically expressedwithin SMC in the subject. The expression vectors can be introduced orreintroduced into a subject (e.g., a human patient) at positions whichallow for the amelioration of SMC-related disease. The subtype-specificSM-MHC promoter derivatives that are selectively active in subsets ofSMC (e.g. vascular versus gastrointestinal SMC, large versus smallarteries, pulmonary versus gastrointestinal SMC, etc.) enable targetedgene expression in specific subtypes of smooth muscle in vivo. Thus,advantages of the targeting methods of the present invention includecomplete SMC specificity, the ability to target specific SMC subsets, asmall size compatible with existing gene delivery methods, and highlevel activity.

For example, the expression vectors of the present invention can beemployed in targeting expression of a therapeutic gene to the specificsubtype of SMC desired (e.g. bronchiolar SMC for treatment of asthma orchronic bronchitis) thereby increasing the efficacy of the therapy andreducing potential side effects due to over-expression in undesiredtissues and cells. In addition, the expression vectors can also be usedin development of animal models of human disease to assist indevelopment of new therapeutic targets. Further, the expression vectorsand targeting methods of the present invention can also be used inidentification and/or selection of smooth muscle cells derived frommulti-potential stem cell populations for purposes of tissuegeneration/regeneration for surgery (e.g. for blood vessel, bladder, orgastrointestinal smooth muscle tissue augmentation-reconstitution).

A. Diseases Amenable to Treatment with Methods of the Present Invention

The present invention provides compositions and methods for targetedgene delivery and expression that can be used to treat a variety ofdiseases and conditions. A large number of major human diseasesincluding systemic hypertension, pulmonary hypertension,atherosclerosis, asthma, coronary artery disease, gastrointestinalabnormalities, reproductive dysfunction, and chronic bronchitis areassociated with abnormal function of the smooth muscle cell (SMC). Aspecific example is to target over-expression of nitric oxide synthase(the enzyme responsible for production of the SMC relaxant nitric oxideor NO from L-arginine) to bronchiolar SMC using our SM-MHC promoterderivative that is active in bronchiolar SMC but inactive in many otherSMC tissues. This targeting would be critical to avoid potentialdeleterious effects of over-expression of NO in other SMC subtypesincluding vascular SMC which might be associated with severe hypotensionand possible death. Similar approaches could be used to target NOsynthase to arteriolar SMC as a means of treating certain forms ofhypertension that are resistant to current therapies.

The present invention also find application in development of animalmodels of disease for purposes of testing potential new drugs/therapies,and/or identifying disease mechanisms. For example, one mightover-express protease enzymes in vascular SMC in large blood vessels asa model to study development of aneurysms and ways to prevent or treatthem. Additional applications of the present invention includedevelopment of gene targeting therapies for promoting formation ofcollateral vessels following tissue ischemia. Methods of the presentinvention can be used in developing ways to promote formation ofcollateral blood vessels in the myocardium following a non-fatal heartattack. For example, the targeting methods of the present invention canbe used to over-express angiogenic substances such as VEGF in thecoronary microcirculation in an ischemic heart region.

The present inventors have identified a molecular mechanism that appearsto be important in mediating repression of SM cell marker genes such asSM-MHC and SM22a that occur when SM cells undergo phenotypic modulationin response to vascular injury. Specifically, a G/C repressor elementwas identified within the promoters of both the SM-MHC and SM22 genes.This repressor element was found to mediates suppression of the activityof these promoters in phenotypically modulated cultured SMC (see, e.g.,Madsen et al., J. Biol. Chem. (1997) 272:6332-6340; and Madsen et al.,J. Biol. Chem. (1997) 272:29842-29851). It was shown that mutation ofthe SM22a G/C element prevented injury-induced down-regulation of thesegenes, but did not affect the tissue selectivity of this promoter. Suchan repressor element can be the target (or “useful”) in SMC genetargeting applications that are associated with phenotypic modulation ofSMC, e.g., post-angioplasty restenosis, intimal SMC withinatherosclerotic lesions, or vascular remodeling in pulmonaryhypertension, etc. For example, the SM-MHC promoter/enhancer of thepresent invention can be used in the context of vascular injury in whichactivity of the wild type SM-MHC promoter is repressed.

B. Targeted Delivery of Therapeutic Agents

Therapeutic agents to be delivered with the targeting methods of thepresent invention include any therapeutic polynucleotide operably linkedto an SM-MHC promoter sequence in an expression vector discussed above.Therapeutic polynucleotides (including those that can be identified withthe screening methods described below) expressed by SM-MHC promotersequences are either active themselves (e.g., antisense and catalyticpolynucleotides) or encode a therapeutic protein.

1. Antisense and Catalytic Ribonucleotides

One type of therapeutic polynucleotide that can be expressed by SM-MHCpromoter sequences is antisense RNA. In such embodiments, the SM-MHCpromoter sequence is operably linked to a polynucleotide which, whentranscribed by cellular polymerases, is capable of binding to targetMRNA. The derivation of an antisense sequence, based upon a cDNAsequence encoding a target protein is described in, for example, Steinand Cohen, Cancer Res 48:2659 (1988) and van der Krol et al.,BioTechniques 6:958 (1988). Antisense oligonucleotides that formtriplexes with a target promoter regions inhibit the activity of thatpromoter, see, e.g., Joseph (1997) Nucleic Acids Res. 25:2182-2188;Alunni-Fabbroni (1996) Biochemistry 35:16361-16369; Olivas (1996)Nucleic Acids Res 24:1758-1764. Alternatively, antisenseoligonucleotides that hybridize to the promoter sequence can be used toinhibit promoter activity.

In addition to antisense polynucleotides, ribozymes can be designed toinhibit expression of target molecules. A ribozyme is an RNA moleculethat catalytically cleaves other RNA molecules. Accordingly, SM-MHCpromoter sequences may be used to express ribozymes specifically insmooth muscle cells by linking a polynucleotide encoding a ribozyme toan SM-MHC promoter sequence. Methods for constructing and usingribozymes to treat smooth muscle cancer in particular are described byDorai et al., Smooth muscle 32:246-58 (1997); Norris et al., Adv Exp MedBiol 465:293-301 (2000). Different kinds of ribozymes have beendescribed, including group I ribozymes, hammerhead ribozymes, hairpinribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al.(1994) Adv. in Pharmacology 25: 289-317 for a general review of theproperties of different ribozymes). The general features of hairpinribozymes are described, e.g., in Hampel et al. (1990) Nucl. Acids Res.18: 299-304; Hampel et al. (1990) European Patent Publication No. 0 360257; U.S. Pat. No. 5,254,678. Methods of preparing are well known tothose of skill in the art (see, e.g., Wong-Staal et al., WO 94/26877;Ojwang et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6340-6344; Yamada etal. (1994) Human Gene Therapy 1: 39-45; Leavitt et al. (1995) Proc.Natl. Acad. Sci. USA 92: 699-703; Leavitt et al. (1994) Human GeneTherapy 5: 1151-120; and Yamada et al. (1994) Virology 205: 121-126).

2. Therapeutic Proteins

A wide variety of therapeutic proteins may be used to treat smoothmuscle diseases. Accordingly, the SM-MHC promoter sequences of thepresent invention may be used to express polynucleotides encodingtherapeutic proteins specifically in smooth muscle cells. Therapeuticproteins may be of prokaryotic, eukaryotic, viral, or synthetic origin.Where the therapeutic protein is not of mammalian origin, the codingsequence of the protein may be modified for maximal mammalian expressionaccording to methods known in the art (e.g., mammalian codon usage andconsensus translation initiation sites).

Therapeutic proteins that can be employed in the targeted gene deliverymethods of the present invention include proteins that kill the cellwhen expressed, such as microbial toxins (Pang, Cancer Gene Ther 7:991-6(2000)) and proteins involved in apoptosis (Li et al., Cancer Res61:186-91 (2001); Schumacher et al., Int J Cancer 91:159-66 (2001); Hyeret al., Mol Ther 2:348-58 (2000); Griffith et al., J Immunol 165:2886-94(2000)). Smooth muscle cells can also be targeted with proteins thatsensitize smooth muscle cells to therapy. Such proteins may function byconverting a prodrug to an active metabolite (e.g., thymidine kinase orcytosine deaminase; for review see Aghi et al., J Gene Med 2: 148-64(2000)), by increasing cell permeability to a therapeutic agent, byrestoring hormonal responsiveness, or by rendering the cell moresensitive to radiotherapy or chemotherapeutics. See, e.g., Suzuki etal., Cancer Res 61:1276-9 (2001); Cowen et al., Clin Cancer Res 6:4402-8(2000); Spitzweg et al., Cancer Res 60:6526-30 (2000); Anello et al., JUrol 164:2173-7 (2000); Fan et al., Cancer Gene Ther 7:1307-14 (2000);Nielsen, Oncol Rep 7:1191-6 (2000); Ayala et al., Hum Pathol 31:866-70(2000); Boland et al., Cancer Res 60:3484-92 (2000). Other proteins thatcan be employed include proteins that inhibit proliferation or act asanti-oncogenes or tumor suppressors (Shirakawa et al., J Gene Med2:426-32 (2000); Tanaka et al., Oncogene 19:5406-12 (2000); Okegawa etal., Cancer Res 60:5031-6 (2000); Allay et al., World J Urol 18:111-20(2000); Steiner et al., Cancer Res 60:4419-25 (2000)), proteins thatinhibit angiogenesis (Jin et al., Cancer Gene Ther 7:1537-42 (2000)) andproteins that induce an immune response, such as cytokines or foreignantigens (Hull et al., Clin Cancer Res 6:4101-9 (2000)). See also U.S.Pat. No. 6,136,792.

C. Delivery System for Targeted Gene Delivery

The expression vectors of the present invention can be transfected intocells for therapeutic purposes in vitro and in vivo. Thesepolynucleotides can be inserted into any of a number of well-knownvectors for the transfection of target cells and organisms as describedbelow. The expression vectors can be delivered in vivo by administrationto an individual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with apolynucleotide (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic polynucleotides can also be administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such polynucleotides areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and viral infection in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu etal., Gene Therapy 1:13-26 (1994)).

Delivery of the gene or genetic material into the cell is the first stepin gene therapy treatment of disease. A large number of delivery methodsare well known to those of skill in the art. Preferably, thepolynucleotides are administered for in vivo or ex vivo gene therapyuses. Non-viral vector delivery systems include DNA plasmids, nakedpolynucleotide, and polynucleotide complexed with a delivery vehiclesuch as a liposome. Viral vector delivery systems include DNA and RNAviruses, which have either episomal or integrated genomes after deliveryto the cell.

Methods of non-viral delivery of polynucleotides include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:polynucleotide conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described in,e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat.No. 4,897,355 and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

The preparation of lipid:polynucleotide complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery ofpolynucleotides take advantage of highly evolved processes for targetinga virus to specific cells in the body and trafficking the viral payloadto the nucleus. Viral vectors can be administered directly to patients(in vivo) or they can be used to treat cells in vitro and the modifiedcells are administered to patients (ex vivo). Conventional viral basedsystems for the delivery of polynucleotides could include retroviral,lentivirus, adenoviral, adeno-associated and herpes simplex virusvectors for gene transfer. Viral vectors are currently the mostefficient and versatile method of gene transfer in target cells andtissues. Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV),and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the polynucleotide ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetpolynucleotides, e.g., in the in vitro production of polynucleotides andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

In particular, a number of viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al, Blood 85:3048-305 (1995); Kohn et al.,Nat. Med. 1:1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci.U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeuticvector used in a gene therapy trial. (Blaese et al., Science 270:475-480(1995)). Transduction efficiencies of 50% or greater have been observedfor MFG-S packaged vectors (Ellem et al., Immunol Immunother.44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997)).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used in transient expression gene therapy, because theycan be produced at high titer and they readily infect a number ofdifferent cell types. Most adenovirus vectors are engineered such that atransgene replaces the Ad E1a, E1b, and E3 genes; subsequently thereplication defective vector is propagated in human 293 cells thatsupply deleted gene function in trans. Ad vectors can transduce multipletypes of tissues in vivo, including nondividing, differentiated cellssuch as those found in the liver, kidney and muscle system tissues.Conventional Ad vectors have a large carrying capacity. An example ofthe use of an Ad vector in a clinical trial involved polynucleotidetherapy for antitumor immunization with intramuscular injection (Stermanet al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the useof adenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 241:5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

D. Pharmaceutical Compositions

The invention provides pharmaceutical compositions that comprise SM-MHCpromoter-containing therapeutic polynucleotides (e.g., oligo- andpoly-nucleotides, expression vectors, gene therapy constructs, etc.)alone or in combination with at least one other agent, such as, e.g., astabilizing compound, diluent, carrier, cell targeting agent, or anotheractive ingredient or agent. The therapeutic agents of the invention maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. Any of these molecules can be administered to a patient alone, orin combination with other agents, drugs or hormones, in pharmaceuticalcompositions where it is mixed with suitable excipient(s), adjuvants,and/or pharmaceutically acceptable carriers. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered (e.g., polynucleotide, protein, modulatory compounds ortransduced cell), as well as by the particular method used to administerthe composition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed., 1989).

Pharmaceutical compositions of the invention include SM-MHCpromoter-containing polynucleotides in an effective amount to achievethe intended purpose. “Therapeutically effective amount” or“pharmacologically effective amount” are well recognized phrases andrefer to that amount of an agent effective to produce the intendedpharmacological result. For example, a therapeutically effective amountis an amount sufficient to treat a disease or condition or amelioratethe symptoms of the disease being treated. The therapeutically effectivedose can be estimated initially either in cell culture assays or in anyappropriate animal model. The animal model is also used to estimateappropriate dosage ranges and routes of administration in humans. Indetermining the effective amount of the vector to be administered, thephysician evaluates circulating plasma levels of the vector, vectortoxicities, progression of the disease, and the production ofanti-vector antibodies. In general, the dose equivalent of a nakedpolynucleotide from a vector is from about 1 μg to 100 μg for a typical70 kilogram patient, and doses of vectors which include a retroviralparticle are calculated to yield an equivalent amount of therapeuticpolynucleotide.

The pharmaceutical compositions of the invention can be administered byany means, such as, e.g., injection, oral administration, inhalation,transdermal, or parenteral application. Methods of parenteral deliveryinclude e.g., topical, intra-arterial (e.g., directly to the tumor),intramuscular (IM), subcutaneous (SC), intramedullary, intrathecal,intraventricular, intravenous (IV), intraperitoneal (IP), or intranasaladministration. Further details on techniques for formulation andadministration may be found in the latest edition of “REMINGTON'SPHARMACEUTICAL SCIENCES” (Maack Publishing Co, Easton Pa.). See also,e.g., PCT publication WO 93/23572.

V. Screening for Modulators of SM-MHC Promoter/Enhancer

The invention also provides constructs, cell lines and methods forscreening for small molecule modulators of SM-MHC promoter/enhanceractivity in vitro and in vivo. Many assays are available that screen forsmall molecule modulators of SM-MHC transcription, including highthroughput assays.

As described in detail in the Examples, results from constructscontaining an SM-MHC promoter/enhancer sequence and a marker gene (inthis example, the lacZ gene) indicated that various motifs of SM-MHCpromoter/enhancer sequence play a role in the SM specificity and subtypespecificity of the SM-MHC promoters. These constructs can be employedfor high throughput screening of modulators of the SM-MHC modulators.Additional cis-acting regulatory elements within an SMCpromoter/enhancer can also be identified as described in the presentinvention.

The present invention also encompasses assays for identifying compoundsthat modulate expression under the SM-MHC promoter sequences. Suchmodulatory compounds are useful in enhancing or inhibiting theexpression of genes transcribed by the SM-MHC promoters, thus providingadditional control and specificity over their expression. Compounds andother substances that modulate expression of the SM-MHCpromoter/enhancer can be screened using in vitro cellular systems. Afterapplying a compound or other substance to the test system, RNA can beextracted from the cells. The level of transcription of a specifictarget gene can be detected using, for example, standard RT-PCRamplification techniques and/or Northern analysis. Alternatively, thelevel of target protein production can be assayed by using antibodiesthat detect the target gene protein. Preferably, the SM-MHC can be fusedto a reporter gene and the expression of the reporter gene can beassessed. Such reporter genes, for which assays are well known to thoseof skill in the art, include, but are not limited to lacZ, βglucoronidase, enhanced green fluorescence protein, etc. See, e.g.,Khodjakov et al., 1997, Cell. Motil. Cytoskeleton, 38:311-317. The levelof expression is compared to a control cell sample which was not exposedto the test compound. The activity of the compounds also can be assayedin vivo using transgenic animals according to the methods described, forexample, in Examples 2-5, below.

Compounds that can be screened for modulation of expression of thetarget gene include, but are not limited to, small inorganic or organicmolecules, peptides, such as peptide hormones analogs, steroid hormones,analogs of such hormones, and other proteins. Compounds thatdown-regulate expression include, but are not limited to,oligonucleotides that are complementary to the 5′-end of the mRNA of theSM-MHC and inhibit transcription by forming triple helix structures, andribozymes or antisense molecules which inhibit translation of the targetgene mRNA. Techniques and strategies for designing such down-regulatingtest compounds are well known to those of skill in the art.

A. Identifying Cis-Acting Elements of SM-MHC Promoter/Enhancer

Multiple cis-elements identified within the first 4.2-kb of 5′-flankingsequence of the SM-MHC promoter are critical for expression in culturedSMC. (White S. L. et al., 1996, J. Biol. Chem., 271:15008-15017; KatohY. et al., 1994, J. Biol. Chem., 269:30538-30545; Wantanabe M. et al.,1996, Circ. Res., 78:978-989; Kallmeier R. C. et al., 1995, J. Biol.Chem., 270:30949-30957; Madsen C. S. et al., 1997, J. Biol. Chem.,272:6332-6340; Madsen C. S. et al., 1997, J. Biol. Chem.,272:29842-29851). The fact that the p4.2-lacZ construct was found to beactive in cultured SMC, but completely inactive in vivo, indicates thatadditional regulatory elements are necessary for expression within thein vivo context. Furthermore, the fact that the p4.2-Intron-lacZconstruct containing approximately 16 kb of the rat SM-MHC genomicregion from −4.2 kb to +11.7 kb was expressed in SMC-tissues withintransgenic mice whereas the p4.2-lacZ construct was inactive, stronglysuggests that the first 11.6 kb region of intron I contains enhancerelements required for expression in vivo but not in cultured SMC.

Additional cis-acting elements of SM-MHC promoter/enhancer can beidentified using methods of molecular genetic analysis well known in theart. For example, the location of cis-regulatory elements within apromoter/enhancer may be identified using methods such as DNase orchemical footprinting (e.g. Meier et al., 1991, Plant Cell 3:309-315) orgel retardation (e.g., Weissenbom & Larson, 1992, J. Biol. Chem.267-6122-6131; Beato, 1989, Cell 56:335-344; Johnson et al., 1989, Ann.Rev. Biochem. 58:799-839). Additionally, resectioning experiments alsomay be employed to define the location of the cis-regulatory elements.For example; a promoter/enhancer containing fragment may be resectedfrom either the 5′ or 3′ end using restriction enzyme or exonucleasedigests.

Another method for identifying transcriptional regulatory motifsinvolves modifying putative cis-acting regulatory subsequences andassessing the change, if any, of the resultant SM-MHC promoter/enhancerto modulate transcription. The modification can be, e.g., one or moreresidue deletions, residue substitution(s), chemical alteration(s) ofnucleotides, and the like. The (modified) promoter can be operablylinked to a transcribable sequence (e.g., reporter genes). The relativeincrease or decrease the modification has on transcriptional rates canbe determined, e.g., by measuring the ability of the unaltered SM-MHCpromoter/enhancer to transcriptionally activate the reporter codingsequence under the same conditions as used to test the modifiedpromoter. An increase or decrease in the ability of the modified SM-MHCpromoter/enhancer to induce transcription as compared to the unmodifiedpromoter construct identifies a cis-acting transcriptional regulatorysequence that is involved in the modulation of SM-MHC promoter/enhanceractivity.

The reporter gene can encode any detectable protein known in the art,e.g., detectable by fluorescence or phosphorescence or by virtue of itspossessing an enzymatic activity. In alternative embodiments, thedetectable protein is firefly luciferase, alpha-glucuronidase,alpha-galactosidase, chloramphenicol acetyl transferase, greenfluorescent protein, enhanced green fluorescent protein, and the humansecreted alkaline phosphatase.

B. Identifying SM-MHC Promoter/Enhancer Trans-Acting TranscriptionalRegulatory Factors

The invention provides means to identify and isolate trans-actingtranscriptional regulatory factors that are involved in modulating theactivity of the SM-MHC promoter/enhancer. Identification of cis-actingmotifs by, e.g., sequence identity comparison, can be a useful initialmeans to identify promoter sequences bound by trans-acting factors. Forexample, as discussed above, the hSM-MHC and rSM-MHC promoter/enhancerscontain a variety of cis-acting motifs (e.g., the CArG motifs and theG/C repressor).

After positive or tentative identification of a cis-acting binding sitein an SM-MHC promoter/enhancer, these sequences are used to isolate thetrans-acting transcriptional regulatory factor(s) by any means known inthe art. In some embodiments, the trans-acting factors are isolatedusing sequence-specific oligonucleotide affinity chromatography, theoligonucleotides comprising SM-MHC promoter sequences of the invention.

Another method tests the ability of the cis-acting elements to bindsoluble polypeptide trans-acting factors isolated from differentcellular compartments, particularly trans-acting factors expressed innuclei. For identification and isolation of factors that stimulatetranscription, cell (e.g. nuclear) extracts from cells that expressSM-MHC are used. Means to conduct these studies are well known in theart (see also Example 5).

Furthermore, as discussed further below, once a cis-acting motif, orelement, is identified, it can be used to identify and isolatetrans-acting factors in a variety of cells and under differentconditions (e.g., cell proliferation versus cell senescence).Accordingly, the invention provides a method for screening fortrans-acting factors that modulate SM-MHC promoter/enhancer activityunder a variety of conditions, developmental states, and cell types(including, e.g., normal versus immortal versus malignant phenotypes).

C. High Throughput Screening of Small Molecule Modulators of SM-MHCPromoter

The invention provides constructs and methods for screening modulators,in a preferred embodiment, small molecule modulators, of SM-MHCpromoter/enhancer activity in vitro and in vivo. The inventionincorporates all assays available to screen for small moleculemodulators of SM-MHC transcription. In a preferred embodiment, highthroughput assays are adapted and used with the SM-MHC promoter/enhancersequences and constructs provided by the invention. See, e.g., Schultz(1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) Mol Divers.3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; Sittampalam(1997) Curr Opin Chem Biol 1:384-91.

One embodiment of the invention provides a method of screening andisolating an SM-MHC promoter/enhancer binding compound by contacting anSM-MHC promoter/enhancer sequence of the invention particularly, anidentified cis-acting regulatory sequence) with a test compound andmeasuring the ability of the test compound to bind the selectedpolynucleotide. The test compound, as discussed above, can be any agentcapable of specifically binding to an SM-MHC promoter/enhancer activity,including compounds available in chemical (e.g., combinatorial)libraries, a cell extract, a nuclear extract, a protein or peptide.

A variety of well-known techniques can be used to identify polypeptideswhich specifically bind to SM-MHC promoter/enhancer sequences, e.g.,mobility shift DNA-binding assays, methylation and uracil interferenceassays, DNase and hydroxy radical footprinting analysis, fluorescencepolarization, and UV crosslinking or chemical cross-linkers. For ageneral overview, see, e.g., Ausubel, supra, (chapter 12, DNA-ProteinInteractions); McLaughlin (1996) Am. J. Hum. Genet. 59:561-569; Tang(1996) Biochemistry 35:8216-8225; Lingner (1996) Proc. Natl. Acad. Sci.USA 93:10712; and Chodosh (1986) Mol. Cell. Biol 6:4723-4733. Where anantibody may already be available or one can be easily generated,co-immunoprecipitation analysis can be used to identify and isolateSM-MHC promoter/enhancer-binding, trans-acting factors. The trans-actingfactor can be characterized by peptide sequence analysis. Onceidentified, the function of the protein can be confirmed by methodsknown in the art, for example, by competition experiments, factordepletion experiments using an antibody specific for the factor, or bycompetition with a mutant factor.

Alternatively, SM-MHC promoter/enhancer-affinity columns can begenerated to screen for potential SM-MHC binding proteins. In avariation of this assay, SM-MHC promoter/enhancer sequence or asubsequences is biotinylated, reacted with a solution suspected ofcontaining a binding protein, and then reacted with a strepavidinaffinity column to isolate the polynucleotide or binding protein complex(see, e.g., Grabowski (1986) Science 233:1294-1299; Chodosh (1986)supra). The promoter-binding protein can then be conventionally elutedand isolated. Mobility shift DNA-protein binding assay usingnondenaturing polyacrylamide gel electrophoresis (PAGE) is an extremelyrapid and sensitive method for detecting specific polypeptide binding toDNA (see, e.g., Chodosh (1986) supra, Carthew (1985) Cell 43:439-448;Trejo (1997) J. Biol. Chem. 272:27411-27421; Bayliss (1997) NucleicAcids Res. 25:3984-3990).

Interference assays and DNase and hydroxy radical footprinting can beused to identify specific residues in the polynucleotide protein-bindingsite, see, e.g., Bi (1997) J. Biol. Chem. 272:26562-26572; Karaoglu(1991) Nucleic Acids Res. 19:5293-5300. Fluorescence polarization is apowerful technique for characterizing macromolecular associations andcan provide equilibrium determinations of protein-DNA andprotein-protein interactions (see, e.g., Lundblad, 1996, Mol.Endocrinol. 10:607-612).

Proteins identified by these techniques can be further separated on thebasis of their size, net surface charge, hydrophobicity and affinity forligands. In addition, antibodies raised against such proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese general methods are well known in the art. See, e.g., Scopes, R.K., Protein Purification: Principles and Practice, 2nd ed., SpringerVerlag, (1987).

The following examples are provided for illustrative purposes and arenot intended to limit the scope of the invention.

EXAMPLES Example 1 General Methods For Analyzing SM-MHC Transgenes

1. Isolation and Cloning of the SM-MHC Promoter/Enhancer

The SM-MHC gene contains a very short untranslated first exon (88 basepairs in rat) that is followed by a greater than 20 kb first intron.Babij P. et al., 1991, Proc. Natl. Acad. Sci., 88: 10676. The cloningand sequence of the 5′-flanking region of the rat SM-MHC gene (−4229 to+88) has been previously reported. White S. L. et al., 1996, J. Biol.Chem., 271:15008-15017; Madsen C. S. et al., 1997, J. Biol. Chem.,272:6332-6340. To obtain 5′-flanking sequences with additional intronicDNA, a rat genomic phage library (Stratagene Corp. La Jolla, Calif.) wasscreened utilizing standard Southern blotting techniques, and α³²P-radiolabeled 45-mer oligonucleotide corresponding to the conserveduntranslated first exon as a probe (nucleotides +14 to +58). One of thepositive recombinant lambda phage clones identified contained anapproximately 16 kb insert (determined by restriction enzyme andsequence analyses) that spanned the SM-MHC gene from −4,216 to +11,795.Identical restriction enzyme patterns between rat genomic DNA andmultiple positive clones revealed that none of the clones identified hadundergone rearrangement.

The nucleotide sequence of the rat clone which was used as the SM-MHCpromoter/enhancer of the present invention is shown in SEQ ID N:21. Theclone spans the rat MHC gene from position −4,216 in relation to thetranscription start site to position +11,795 downstream of thetranscription start site, thus, containing about 16,011 base pairs intotal. Furthermore, since the first exon of the rat MHC gene is 88 basepairs in length, the clone extends to +11,707 base pairs within thefirst intron.

Although the instant example describes the cloning and isolation of therat SM-MHC promoter/enhancer, key regulatory regions within thispolynucleotide sequence are known to be conserved across all speciesthat express the gene. Thus, the instant invention encompasses not onlythe rat SM-MHC, but also the SM-MHC of other mammals, including, but notlimited to, humans, rabbits and mice. The full length human SM-MHC genesequence has previously been deposited with the Institute for GenomicResearch in Rockville, Md., and is assigned Ace. No. U91 323 and NID No.G233 5056. It can be accessed athttp://www.ncbi.nlm.nih.gov/htbin-post/Entrezlquery?db=n_d. Thissequence is hereby incorporated by reference in its entirety. Based upona comparison of the human and rat SM-MHC gene sequences, FIG. 11 showsthe high degree of homology that exists between the rat and human genes.In fact, as shown in FIG. 11, critical regulatory sequences are 100%conserved within the genes. Furthermore, it has previously been shownthat similar regulatory sequences are conserved in the rabbit and mousegenes for SM-MHC. See, Madsen et al., 1997, J. Biol. Chem. 272:6332.

2. Construction of the SM-MHC-LacZ Reporters

To facilitate removal of pBS plasmid DNA from the pBS-lacZ vector, thepBS-lacZ vector was modified by inserting Not I restriction enzymerecognition sites at the HindIII and EcoRI sites located at the bordersof the pBS vector sequence. Two SM-MHC-lacZ reporter genes wereconstructed for the generation of transgenic mice. One construct (p4.2lacZ) was created by ligating about a 4.3 kb BgIII fragment thatextended from −4220 to +88 into a unique BamHI site of the pBS-lac-Zvector, and the other construct tested (p4.2-Intron-lacZ) was generatedby subcloning an approximately 16 kb SaII fragment that extended from−4229 to about +11,700 into the SaII site of the pBS-lacZ vector. Tofacilitate splicing of the p4.2-Intron-lacZ construct, a syntheticsplice acceptor site was ligated into the KpnI site of the pBS-lacZvector prior to insertion of the SM-MHC DNA fragment. The location ofthe KpnI site, between the SaII site and the lacZ-gene, allowed for thecorrect positioning of the splice acceptor site at the +11,700 end ofthe SM-MHC intron. The proper construction of each SM-MHC-lacZ chimericplasmid was verified by sequencing and restriction enzyme analyses. Asan additional precaution against cloning artifacts, both transgenicconstructs were tested for lacZ expression in transient transfectionassays in cultured rat aortic SMC using a method that was previouslydescribed. Madsen C. S. et al., 1997, J. Biol. Chem., 272:6332-634

3. Generation and Analysis of Transgenic Mice

Plasmid constructs p4.2-lacZ and p4.2-Intron-lacZ were tested for SM-MHCpromoter activity in transgenic mice following removal of the pBS vectorDNA through NotI digestion and subsequent agarose gel purification.Transgenic mice were generated using standard methods (Li L. et al,1996, J. Cell. Biol., 132:849-859; Gordon J. W. et al., 1981, Science, 214:1244-1246) either commercially (DNX, Princeton, N.J.) or within theTransgenic Core Facility at The University of Virginia. Transgenic micewere either sacrificed and analyzed during embryological development(transient transgenics), or were utilized to establish breeding-founderlines (stable transgenics). Transgene presence was assayed by thepolymerase chain reaction using genomic DNA purified from eitherplacental tissue (embryonic mice) or from tail clips (adult mice)according to the method of Vemet M. et al., 1993, Methods Enzymol.225:434-451. Transgene expression and histological analyses were done asdescribed previously. Li L. et al., 1996, J. Cell. Biol., 132:849-859;Cheng T. C. et al., 1993, Science, 261:215-218. In order to determinepossible positional effects of transgene insertional sites on transgeneexpression, multiple independent founder lines were analyzed for eachtransgene construct.

4. SM-MHC Immunohistochemistry

Various smooth muscle containing tissues were collected from 5-6 weekold transgenic mice and fixed overnight in methacam (60% methanol, 30%chloroform, 10% glacial acetic acid). Tissues were subsequentlydehydrated through a graded series of methanol dilutions. Fixed,dehydrated tissues were prepared for paraffin embedding by incubation in100% xylene. Tissue was then infiltrated by incubation through a seriesof xylene:paraffin (3:1, 1:1, 1:3) solutions, and two final incubationsin 100% paraffin prior to embedding in 100% paraffin. Serial sections (6μm) were placed on uncoated slides, and then dried for approximately 45minutes on a slide warmer set at 40° C. Sections were cleared inmultiple washes of 100% xylene, and re-hydrated through a graded ethanolseries to a final incubation in phosphate buffered saline (PBS).Endogenous peroxidase activity was quenched by incubating slides inmethanol containing 0.3% hydrogen peroxide for 30 min. Slides weresubsequently rehydrated in PBS and blocked in a 1:50 solution of normalgoat serum made up in PBS. Sections were then incubated with the primaryantibody for 1 hr and washed with 3 changes of PBS. Detection of primaryantibody was performed using a Vectastain ABC Kit according to theinstructions of the manufacturer with diaminobenzidine (DAB) as thechromagen (Vector Laboratories, Burlingame, Calif.).

Antibodies:

Several different SM-MHC antibodies were employed. These included amonoclonal antibody designated 9A9 which has been previouslycharacterized (Price R. J. et al., 1994, Circ. Res., 75:520-527) thatshows reactivity with the SM-1 and SM-2 isoforms of SM-MHC but whichshows no reactivity with non-muscle myosin heavy chains or otherproteins. However, whereas this antibody showed some reactivity withmouse SM-MHC isoforms in Western analyses, it reacted very poorly withmouse SM-MHC in fixed tissues. In addition, although a polyclonal SM-MHCpeptide antibody provided by Nagai R. et al, 1989, J. Biol. Chem.,264:9734-973 7, showed complete specificity for SM-MHC isoforms inWestern analyses of smooth muscle tissues from multiple species, itshowed little or no reactivity with mouse SM-MHC isoforms. To circumventthese limitations, a rabbit anti-chicken gizzard SM-MHC polyclonalantibody was employed. The rabbit anti-chicken gizzard SM-MHC antibodywas made by immunization of rabbits with partially purified gizzardSM-MHC as described by Groschel-Stewart, 1976, Histochemistry46:229-236. However, based on Western analyses, it was determined thatthis antibody showed reactivity with both SM-1 and SM-2 MHC, as well aswith non-muscle myosin B (or SMEMB), as did a number of other “smoothmuscle myosin” antibodies tested, including one from Sigma [designatedhSM-V] (Frid M. G. et al., 1993, J. Vasc. Res., ;30:279-292) and onefrom R. S. Adelstein (Schneider M. D. et al., 1985, J. Cell. Biol.,101:66). As such, staining with these antibodies in tissues that expressboth SMEMB and SM-MHC is equivocal. However, adult mouse aortic SMC,like those in other species (Rovner A. S. et al., 1986, J. Biol. Chem.,261: 14740-14745; Rovner A. S. et al., 1986, Am. J Physiol.,250:c861-c870; Phillips C. L. et al., 1995, J. Muscle Res. & CellMotility, 16:379-389) were not found to express SMEMB based on Westernanalyses. The rabbit anti-chicken gizzard SM-MHC polyclonal antibody wasused at a concentration of approximately 20 μg/ml in PBS. Biotinylatedgoat anti-rabbit secondary antibodies were purchased from VectorLaboratories (Burlingame, Calif.) and used at a concentration of 10μg/ml in PBS. Appropriate Western analyses, and immunohistologicalcontrols were performed to assess specificity, including exclusion ofprimary antibody, and use of control non-immune rabbit serum.

5. Chromatin Immunoprecipitation

L6 rat skeletal myoblasts were cultured in α-minimal essential medium(Lifetechnologies) supplemented with 2% FBS for 7 days to induce myotubeformation. L6 myotubes, L6 myoblasts, Rat1 fibroblasts, and rat aorticSMCs in 100 mm dished were fixed directly by adding 280 μl of 37%formaldehyde to 10 ml of culture media and incubating at 37° C. for 10min. The fixed cells were harvested and prepared for immunoprecipitationusing the protocol of ChIP assay kit (Upstate Biotechnology) with minormodifications. A quarter of the sample was precleared with salmon spermDNA/protein A agarose (Upstate) and subsequently incubated with either 2μl of anti-SRF antibody (Santa Cruz Biotechnology) or no antibody at 4°C. over night. Chromatin samples were immunoprecipitated using salmonsperm DNA/protein A (Upstate). Samples were washed two times with 1 mlof wash buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl,pH 8.1, 150 mM NaCl), once with wash buffer B (0.25 M LiCl, 0.5% NP-40,0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and twotimes with TE. Immune complexes were eluted and subsequentlyreverse-crosslinked and purified by phenol/chloroform extraction.Ethanol precipitated DNA pellets were redissolved in 40 μl of TE buffer.The supernatant of an immunoprecipitation reaction done in the absenceof SRF antibody was purified and used as a control to show total inputDNA. The supernatant DNA was diluted 1:100 prior to PCR. One μl of eachsample was subjected to PCR amplification. PCR analysis was carried outusing primers from different regions of the SM-MHC gene and promoterregions of a number of control genes that are silent in SMCs (skeletalα-actin, insulin and β-globin). The sequences of PCR primers are shownin table 1. Following 32 (all primer sets except insulin) or 35 cycles(insulin) of amplification, PCR products were run on 2% agarose gels andanalyzed by GelStar (BMA) staining. As additional controls, the promoterregions of the genes either silent or lacking CArG elements were alsoamplified by PCR. The PCR samples of these promoters showed a low levelof background chromatin immunoprecipitation. The sequences of the PCRprimers were the following;

insulin, 5′-GCCAAAACTCTAGGGACTTTAGGAAGGATG-3′ (SEQ ID NO:10),5′-GCCGGGCAACCTCCAGTGCCAAGGTCTGAAGATC-3′ (SEQ ID NO:11); β-globin,5′-CAGCGTTTTCTTCAGAGGGAGTACCCAGAG-3′ (SEQ ID NO:12),5′-TCAGAAGCAAATGTGAGGAGCGACTGATCC-3′ (SEQ ID NO:13); skeletal α-actin,5′-CAGGCTGAGAAGCAGCCGAAGGGACTCTAG-3′ (SEQ ID NO:14),5′-ACCTCCACCCTACCTGCTGCTCTGACTCTG-3′ (SEQ ID NO:15); SM-MHC -4000,5′-ATGTCAGATGTCCTCTCACTGCTTTATTCC-3′ (SEQ IDNO:21),5′-AGCAAACAGCTTTAAATACGTATTGGCTTC-3′ (SEQ ID NO:22); 5′-CArG,5′-CTGGAGCTCTTATTAGTACTGGGGTCCC-3′ (SEQ ID NO:18),5′-ACTCAGGCCATAAAAGGAAGTCGAGGCAGAGTTGG-3′ (SEQ ID NO:19); intronic CArG,5′-GGC CAAGCCACCCTGGAGAAACCTGGAC-3′ (SEQ ID NO:20),5′-CCCAGAACTCAAGCCAGTCAGGCTGCATCG-3′ (SEQ ID NO:23). Due to therelatively low resolution of ChIP method, we designed the PCR primersfor the 5′-flanking CArG region for amplification of the regioncontaining both CArG1 and CArG2.

Example 2 SMC-Specific Expression in Transgenic Mice: IndispensableElements in SM-MHC First Intron

It has previously been reported that an SM-MHC promoter DNA fragmentextending from −4220 to +88 was capable of directing high-levelexpression in cultured rat aortic SMC. Madsen C. S. et al., 1997, J.Biol. Chem., 272:6332-6340. When tested in bovine endothelial cells, L6myoblasts and L6 myotubes, the activity of this construct was determinedto be negligible. To determine if this same promoter/DNA fragment wascapable of directing SMC-specific expression in vivo, this fragment wassub-cloned into a pBS-lacZ reporter gene construct (p4.2-lacZ) andtested for activity in transgenic mice. Thirteen independent transienttransgenic mice harboring the p4.2-lacZ transgene were generated andanalyzed for lacZ expression at multiple embryological stages rangingfrom embryonic day (“E”) 13.5 to 19.5. No transgene expression wasdetected in any of the transgenic mice. These data show that, incontrast to activity levels observed for cultured SMC, the SM-MHCpromoter fragment present within the p4.2-lacZ construct did not containsufficient DNA for directing SMC-specific expression in transgenic mice.

It is well documented that cis-elements important for gene expressioncan be found outside the 5′-flanking region. Furthermore, they can befound within intronic regions. Because 4.2 kb of 5′-flanking DNA wasfound to be insufficient for expression in vivo, a larger construct withadded intronic sequences was tested. A rat genomic phage library wasscreened and one recombinant clone was identified whose insert contained4216 bp of 5′ flanking region, 88 bp of the first exon, which isuntranslated sequence, and an additional 11,795 base pairs of firstintronic sequence (total span: −4,216 to +11,795). This fragment, whichwas essentially identical to the p4.2-lacZ construct with respect to the5′-flanking sequence and with respect to the presence of the 88 bp of 5′untranslated sequence, was isolated from the lambda phage by SaIIdigestion and sub-cloned into the pBS-lacZ vector to create theSM-MHC-reporter gene plasmid p4.2-Intron-lacZ.

The reporter gene p4.2-Intron-lacZ was used to generate four independenttransgenic mice; one mouse was sacrificed at E13.5 for transgeneexpression analysis, and the other three were established as stabletransgenic founder lines (designated as 2282, 2642 and 2820) that wereutilized for analysis of transgene expression throughout embryologicaldevelopment and early adulthood. Analysis of adult mice generated fromthe three stable founder lines showed that lacZ transgene expression wasessentially identical between the three founders and completelyrestricted to smooth muscle (FIGS. 1, 2 and 12). Gross examination ofthe heart and lung region excised from a 5 week-old p4.2-Intron-lacZmouse revealed that transgene expression was present in the descendingthoracic aorta, coronary arteries, trachea and bronchi (FIG. 12, PanelA). Transgene expression was not detected in any non-smooth muscletissues in this region, such as heart muscle and lung tissue. Of note,transgene expression also was not detected in several smooth musclecontaining tissues in this region including the esophagus and branchesof the pulmonary artery, although expression was seen in the pulmonaryartery outflow tract. Transgene expression was readily detectable in themajor branches of the coronary arterial tree including the left andright coronary arteries (FIG. 12, Panel B), as well as the smallcoronary arteries and arterioles (FIG. 12, Panel D) of 5-6 week oldtransgenic mice. However, no lacZ expression could be detected in any ofthe coronary veins (FIG. 12, Panels B and D; and FIG. 13, Panel C).Transgene expression also was readily detected in the descendingthoracic aorta, and intercostal arteries (FIG. 12, Panel C), as well asthroughout blood vessels in the extremities and main body trunk,including small arteries, arterioles and veins such as the mesenteryvessels (FIG. 12, Panel E). Expression of the lacZ transgene was readilydetectable also in the visceral smooth muscle of the intestine (FIG. 12,Panel F), the ureter and bladder (FIG. 12, Panel G), the stomach (FIG.12, Panel H) and the uterus and gallbladder. Thus, these initialanalyses demonstrated that the p4.2-Intron-lacZ construct containedsufficient DNA for expression in all SMC tissue types, although certainSMC tissues were negative, at least in 5-6 week old animals. Moreover,certain smooth muscle tissues such as the aorta (FIG. 12, Panel C),intercostal arteries (FIG. 12, Panel C), jejunum (FIG. 12, Panel F) andstomach (FIG. 12, Panel H) clearly showed a mosaic pattern of transgeneexpression that was visible even at the gross tissue level.

To assess transgene expression at the cellular level, histologicalanalyses of lacZ reporter expressions were performed (see, e.g., FIGS. 3and 13). Results of these studies further demonstrated that transgeneexpression was highly restrictive to smooth muscle. For example,analysis of the bladder and airway smooth muscle (FIG. 13, Panel A)showed that transgene expression was highly specific and appeared to bepresent in virtually all SMC located within these tissues. Likewise, SMCwithin many smooth muscle tissues including the aorta (FIG. 13, PanelB), coronary vessels (FIG. 13, Panel C), the intestine (FIG. 13, PanelD), stomach and many smaller blood vessels including small arteries,arterioles, veins, and venules (FIG. 13, Panels E and F) showed clearevidence of expression of the transgene within SMC, although someheterogeneity of expression was evident between adjacent cells.

Taken together, these results indicate that although the p42-Intron-lacZtransgene exhibited SMC-specific activity and was expressed in all majorSMC types, it exhibited differences in activity in subsets of SMC bothwithin and between different adult SMC tissues. Nevertheless, expressionof the p4.2-Intron-lacZ transgene was present only in SMC, and not inany non-SMC.

Example 3 Transgene Expression in the Developing Embryo

To determine if expression of the p4.2-Intron-lacZ transgene resembledthe developmental expression pattern of the endogenous SM-MHC gene,embryos from the three stable founder lines were obtained at variousstages throughout development [embryonic day E10.5 through E19.5] andanalyzed for lacZ expression. Additionally, one transient founder wasgenerated and analyzed for transgene expression at E13.5. With theexception of transient expression in the heart (B 12.5 to E17.5) of oneof the stable lines which was localized to the myocardium, transgeneexpression patterns were essentially identical in all four independenttransgenic lines (i.e. one transient transgenic mouse and three stablefounder lines), and restricted to SMC. Transgene expression patterns ofembryos derived from stable founder lines 2282, 2642 and 2820 arepresented in FIGS. 14 and 15. The earliest developmental stage at whichtransgene expression could be detected was E12.5, where lacZ expressionwas readily identified in the trachea and bronchi (FIG. 14, Panels A andB). By E14.5, transgene expression was detectable in the bronchi,intestine, stomach, trachea and the aorta as well as a few other vesselsthroughout the embryo (FIG. 14, Panel C). Of particular interest,although transgene expression was virtually absent in the esophagus inthe adult (FIG. 12, Panel H), its expression was clearly evident inembryos. At E16.5 transgene expression was more pronounced in the aortathan at earlier developmental time points, although it had a variegatedand less intense appearance relative to other smooth muscle tissues(FIG. 14, Panel D). Additionally, the frequency of vessels that werepositive for transgene expression was higher in peripheral vessels, andparticularly those located in the extremities of the animal.

One of the most notable differences between the E16.5 and E19.5 embryoswas a marked increase in the frequency of vessels that stained positivefor lacZ expression (FIG. 15). However, lacZ expression remainedundetectable in a number of vessels. Especially conspicuous was thegeneral absence of expression in the large blood vessels in the head andneck region including the internal and external carotid arteries, thejugular vein and the cerebral arteries and veins. However, many smallersized blood vessels were positive for transgene expression in the headand neck region. Transgene expression was readily detectable also inmany other arteries and veins throughout the body including the iliacs(FIG. 15, Panel D), the caudal artery and vein, the femoral artery, theumbilical artery and vein, the ulnar and radial arteries and superficialarterioles and venules within the musculature of the thoracic cage (FIG.15).

Although expression levels in these types of studies are notquantitative, it is worth noting that levels of lacZ staining within theaorta did not appear to be as intense as compared to many other bloodvessels and visceral smooth muscle tissues. In summary, results of theseembryological studies support the data gathered from analysis oftransgene expression in juvenile and adult mice, and show thatp4.2-Intron-lacZ contains sufficient DNA for directing SMC-specificexpression in all SMC-tissue types. However, results leave open thepossibility that additional genomic regions may be required for SM-MFICexpression in some subsets of SMC. Nevertheless, these resultsdemonstrate that the p4.2 Intron-lacZ transgene is capable of conferringSMC-specific gene expression in vivo.

Example 4 Multiple CArG Elements Define SM-Subtype Specificity of SM-MHCIn Vivo

1. Plasmids Construction and Transfection

Mutant transgenic constructs of SM-MHC CArG elements were made in thecontext of −4200 to +11600 promoter/intron LacZ transgene (SM-MHC4.2+intron-LacZ plasmid; Madsen et al., Circ. Res. 82:908-17, 1998). Forsimplicity, this construct is referred to as SM-MHC −4200/+11600 LacZ inthis paper. Site-directed mutagenesis was performed on small fragmentssubcloned in pBluescript II using GeneEditor (Stratagene). The integrityof mutated fragments was confirmed by sequencing, and the fragments weresubcloned back into the parental plasmid. The resultant mutanttransgenic plasmids were tested for integrity by sequencing andrestriction enzyme mapping. To minimize the possibility of errors in DNAamplification, at least two independently constructed clones were testedfor activity in cultured SMCs. Mutant sequences are the following:CArG1, ttCCTTTTATGG (SEQ ID NO:1) to ggATCCTATGG (SEQ ID NO:2); CArG2,CCTTTTTGGG (SEQ ID NO:3) to ATCCTTTGGG (SEQ ID NO:4); intromc CArG,CCTTGTATGG (SEQ ID NO:5) to AGGCCTATGG (SEQ ID NO:6).

The minimal thymidine kinase promoter taken from pBLCAT5 (Boshart etal., Gene 110:129-30, 1992) was subcloned into pAUG LacZ. Subsequently aBstXI/BglI (+1447 to +1673) fragment of the SM-MHC first intron wassubcloned into the TK LacZ vector so that the fragment was repeatedthree times upstream of the TK promoter (3xICR-TK LacZ).

Transfection of the plasmids was performed using DOTAP (Roche) asdescribed previously (Madsen et al., J. Biol. Chem. 272:6332-40, 1997).At least two independent clones were used for transfection and: thetransfection of each plasmid was done at least in duplicate. Reporteractivity was assayed by using ONPG as a substrate (Manabe et al.,Biochem, Biophys. Res. Commun. 239:598-605, 1997). The activity wasnormalized to the protein concentration of each cell lysate as measuredby DC protein assay kit (BioRad). The endogenous β-galactosidaseactivity was determined by transfecting a nonfunctional DNA (pBluescriptII) and was subtracted from the measured activity of each construct.Subsequently the activity was normalized to that of promoterlessconstruct, pAUG LacZ. One-way analysis of variance followed byBonferroni method was used for data analysis. Values of p<0.05 wereconsidered statitically significant.

Transgenic mice were used to establish breeding founder lines.Transgenic mice were generated and analyzed as described above inExample 1. To determine possible positional effects of transgeneinsertional sites on transgene expression, multiple independent founderlines were analyzed for each transgene construct.

2. Preparation of Nuclear Extracts and Electrophoretic Mobility ShiftAssays (EMSAs)

Transgenic mice were used to establish breeding founder lines.Transgenic mice were generated and analyzed as described above inExample 1. To determine possible positional effects of transgeneinsertional sites on transgene expression, multiple independent founderlines were analyzed for each transgene construct.

Preparation of nuclear extracts from cultured SMCs was performed asdescribed in Madsen (1997). Nuclear extracts from rat tissues wereprepared as described previously (Dignam et al., Nucleic Acids Res.11:1475-89, 1983) with the following modifications. In brief, tissueswere taken from male Sprague-Dawley rats. Non-SMC layers were removedfrom the aorta, stomach, and bladder and tissues were immediately frozenin liquid nitrogen. The frozen tissues were powdered and washed oncewith modified buffer A (10 mM HEPES, pH 7.9, 13 mM KCl, 0.1 mM EDTA, 0.5mM DTT, 0.05% NP-40) with Complete EDTA-free protease inhibitor (Roche).The samples were resuspended in 10 ml of buffer A and incubated on icefor 5 min. The samples were centrifuged and resuspended in a 10 packedcell volume of buffer A. NP-40 was added to the final concentration of0.3%. The samples were then homogenized using a Dounce homogenizer.Disruption of cell membranes was confirmed by microscopic observation.The centrifuged samples were resuspended in modified buffer C (20 mMHEPES, pH 7.9, 420 mM NaCl, 0.2 MM EDTA, 25% glycerol, 0.5 mM DTT, 0.01%NP-40, Complete EDTA-free) and incubated on ice with gentle agitationfor 30 min. Cell debris was removed by centrifugation. The samples werechanged for buffer and enriched using Ultrafree-4 concentrator(Millipore).

The sequences of sense strands of EMSA probes were the following; CArG1,5′-gacttccttttatggcctga-3′(SEQ ID NO:7); CArG2,5′-cctggcctttttgggttgtt-3′(SEQ ID NO:8); intronic CArG,5′-catgcccttgtatggtagtg-3′ (SEQ ID NO:9); EMSAs were performed asdescribed in Manabe et al., supra. In brief, 20 kcpm of ³²P-labeledprobe was incubated with nuclear extracts in 20 μl of binding buffer (10mM Tris-HCl (pH7.5), 50 mM NaCl, 0.5 mM DTT, 10% glycerol, and 0.05%NP-40) with 0.25 μg of poly (dI-dC) (dI-dC). Reactions were incubated onice for 20 min. For supershift assays, 1 μl of anti-SRF antibody wasadded after the 20-min incubation period and the reactions wereincubated for an additional 10 min. The reactions were run on 5%polyacrylamide gels.

3. The SM-MHC Contains Conserved CArG Elements Required for MaximalPromoter Activity in Cultured SMCs

We first examined transcriptional activity of the first intron incultured rat aortic SMCs by using a series of 3′-deletion constructs asa means to identify putative cis-regulatory elements that wouldsubsequently be tested in vivo in transgenic mice. Deletion of theregion from +2491 to +417 decreased activity significantly (56-fold vs.17-fold activity over pAUG LacZ). To identify possible cis-elements inthe region +2491 to +417, we further analyzed this region using finerdeletion mutants. A series of finer deletion mutants was constructed inthe context of 1346 bp of the 5′-flanking region and transfected intocultured SMCs (FIG. 18A). Significant reductions in reporter activitywere observed when the sequence from +1617 to +1586 was deleted (FIG.18A). Within this region there is a CArG-like element at +1599 that isalso present at an equivalent region in the human SM-MHC intron (FIG.18B).

We have previously identified two CArG elements in the 5′-flankingsequence of the SM-MHC gene that are functional in the context of −1346to +88 region in cultured SMCs (Madsen et al., J. Biol. Chem.272:29842-51, 1997; and Madsen et al., J. Biol. Chem. 272:6332-40,1997). However, since the 5′-flanking sequence alone is completelyinactive in SMC in vivo in transgenic mice, it is critical to re-testthe functionality of these cis-elements within the context of the−4200/+11600 LacZ construct previously shown to be active in SMCs invivo (Madsen et al., 1998, supra). To ensure the efficacy of mutationsin abrogating transcription factor binding, we first performed a seriesof EMSA experiments with each CArG element. Consistent with our previousresults, both CArG1 and CArG2 probes bound SRF in nuclear extractsprepared from cultured SMCs (FIG. 19, lanes 21 and 22). In addition, asexpected based on sequence analysis, the SM-MHC intronic CArG alsoexhibited SRF binding (FIG. 19, lanes 23, complexes A and B). Mutationsof each CArG element completely abolished SRF binding activity in EMSAs.We then tested the effects of these same CArG mutations ontranscriptional activity of the −4200/+11600 LacZ SM-MHC construct incultured SMCs. The mutations of CArG1, CArG2, and intronic CArG reducedreporter activity by 46%, 49%, and 74%, respectively.

4. The Intronic CArG Confers SM-Type-Selective Transcription In Vivo

The data of transgenic mouse experiments clearly demonstrated that theintronic CArG element was necessary for transcription of the SM-MHC genein the large arteries in vivo. As shown in FIG. 18B, we found that theregion containing the intronic CArG is highly conserved between the ratand human genes. To test if the intronic region could work as a distincttranscriptional regulatory module in vivo, three copies of the 227-bpsequence containing the highly homologous region (+1447 to +1673) werecloned in tandem 5′ to a minimal thymidine kinase (TK) promoter LacZconstruct (3xICR-TK LacZ). The construct showed very high activity(13.7-fold activity over the minimal TK LacZ construct) in culturedSMCs. This construct was used to produce transgenic mice. In one founderline (line 7240) among four founder lines, very strong reporterexpression was observed in vascular SMCs (see, FIG. 25). Another line(line 7249) also showed expression in SMCs although not as strong. Theother two lines were negative for staining. Although two founder linesdid not express the 3xICR-TK LacZ transgene; it seems unlikely that thecell restricted activity observed in lines 7240 and 7249 was due solelyto locus dependent activation associate with the site of transgeneinsertion. Rather results support that the 227-bp intronic sequence candirect transcription at least in some SMCs in vivo when coupled with aminimal TK promoter.

In line 7240, reporter expression was particularly prominent in thelarge arteries including the aorta, carotid, and pulmonary arteries(FIG. 25, Panels A, B, F, G). Reporter expression was also strong inintermediate size arteries (FIG. 25, F). Transgene expression in smallerarteries was relatively weaker than that in large arteries and not allthe smaller arteries were stained positive. Reporter expression was alsoobserved in large veins including the vena cava (FIG. 25, F). While theexpression in vascular SMCs was very strong, transgene expression wasvery weak in visceral SMCs. Only few cells were stained positive in thestomach, intestine, and bladder (FIG. 25, C-E). Interestingly, strongreporter expression was also observed in the heart and skeletal muscle.In the heart, while cardiac muscle cells were stained positive forβ-galactosidase expression, no transgene expression was observed in SMCsin coronary vessels (FIG. 25, H). Various skeletal muscle cells alsoexpressed the transgene (FIG. 25, I) The data provide evidence that theconserved region containing the intronic CArG is capable of drivingtranscription in subsets of SMCs in vivo but lacks the completeSMC-specificity seen with the endogenous SM-MHC gene and the−4200/+11600 LacZ transgene.

5. Differential Requirements of the CArG Elements in SMC-Subtypes InVivo

In order to examine the functional roles of the CArG elements in vivo,the −4200/+11600 LacZ SM-MHC CArG mutant constructs were used togenerate transgenic mice. The expression patterns observed aresummarized in Table 1. Mutation of CArG1 resulted in abrogation of LacZexpression in all SM tissues in all three independent transgenic founderlines analyzed. In contrast, all three transgenic founder lines of thewild-type SM-MHC −4200/+11600 LacZ construct showed reporter expressionin virtually all SMC tissues (FIG. 20). These data clearly demonstratethat CArG1 was required for expression of the SM-MHC gene in vivo in allSM tissues.

Mutation of CArG2 resulted in differential reductions in reporteractivity in SM tissues. LacZ expression in the gastrointestinal (GI)tract was decreased but was easily detectable in adult mice (FIG. 20, I,M vs. K, O). Expression in the bladder was similar to that observed inwild-type mice (FIG. 20, Q vs. S). No expression was observed in largeblood vessels including the aorta, pulmonary, coronary, carotid, celiac,and femoral arteries, and the vena cava (FIG. 20, A, E vs. C, G).However, very weak reporter expression was observed in smaller arteriesincluding small mesenteric arteries (data not shown). Mutation of CArG2also virtually abolished expression in the trachea and bronchi (FIG. 20,E vs. G).

TABLE 1 Summary of report gene expression in SM-MHC LacZ transgenic miceLacZ positive lines/founder coronary mesenteric construct lines aortaartery artery vena cava airways stomach intestine bladder −4200/+116003/3 ++ ++ ++ ++ ++ ++ ++ ++ LacZ CArG1 mutant 0/3 − − − − − − − − CArG2mutant 3/5 − − ± − ± + + + intronic CArG 4/4 − + ++ ++ ++ ++ ++ ++mutant 3xICR-TK 2/4 +++ − ++ ++ ± ± ± ± LacZ

Mutation of the intronic CArG resulted in a vascular SMC-specificphenotype. Reporter expression in the GI tract, urinary tract andairways was equivalent to that of wild-type transgenic mice in adultsand embryos of four independent intronic CArG mutant lines (FIG. 20,Panels B, I, M, Q vs. H, L, P, T). Expression in veins was alsoequivalent to that of the wild-type. However, expression in largearteries including the aorta, common carotid arteries and the maintrunks of subclavian arteries was completely silenced in all lines (FIG.20, A, E vs. D, H). Interestingly, the small branching arteries from thethoracic aorta including the intercostal arteries showed transgeneexpression equivalent to that of the wild-type (FIG. 21, C, D). In thecarotid arteries no expression was observed in the proximal portion(FIG. 21, E), whereas in the distal common carotid arteries a few cellswere stained positive, and the internal and external carotid arterieswere strongly stained. Strong expression of the intronic CArG mutant wasalso observed in arteries in the head including the basilar artery,arteries of Willis ring, and cerebral arteries (data not shown).Reporter expression was not detected in the abdominal aorta, whereas thebranching arteries from the abdominal aorta including the celiac, renal,and adrenal arteries were stained strongly positive (FIG. 21, A vs. B).Indeed, the abrupt transition in expression from non-detectable to ahigh level between the conduit arteries and branch arteries was quiteremarkable (FIG. 21, B, F). Histological sectioning of blood vessels inthe abdomen clearly showed selective abrogation of reporter expressionin the aorta in intronic CArG mutant transgenic mice (FIG. 21, G-J). Inthe common iliac arteries expression was barely detectable, whereasexpression was strong in their branches including the femoral arteries(FIG. 21, F).

Transgene expression in the coronary arteries was somewhat varied amongthe intronic CArG mutant lines presumably due to positional effects oftransgene insertion sites. In two lines some expression was detectablein the coronary arteries, while little or no expression was observed inthe other lines. However, even in the former two lines, overalltransgene expression was clearly much weaker than that of the wild-typetransgenic lines. Positive staining was restricted within the maintrunks and a few major branches in the intronic CArG mutants, while inthe wild-type expression was detectable in smaller branches. However,due to the qualitative nature of β-galactosidase staining andvariability in expression level among the lines we could not concludethe extent of necessity of the intronic CArG in the coronary arteries.

Similarly, expression in the pulmonary arteries and veins was varied inmice containing the mutant intronic CArG transgene. Two lines, whichshowed transgene expression in the coronary arteries, had detectabletransgene expression in the pulmonary blood vessels, whereas the otherlines showed no expression. Even in two expressing lines, transgeneexpression was very weak, which made the staining of the lung looksparse as compared with that of the lung of wild-type mice as depictedin FIG. 20. However, microscopically some SMCs in the pulmonary bloodvessels were stained positive (data not shown). Expression of thewild-type transgene in the pulmonary vessels was also somewhat variedamong transgenic lines and the expression level, especially that in thepulmonary veins, was generally weak as compared with other vascularbeds. Given the variability and weakness of transgene expression in thepulmonary circulation, no definite conclusions can be made regarding therole of the intronic CArG in pulmonary blood vessels, although resultsshowing no expression in two founder lines suggests that it may havesome function. Reporter expression in intronic CArG mutant transgenicmice showed that the intronic CArG was indispensable for transgeneexpression in large arteries, while it was dispensable in smallerarteries, veins and visceral SMCs. The large arteries that absolutelyrequired the intronic CArG largely fit the classification of elasticartery.

The SM-MHC gene is a marker of later stage SM differentiation, andexpression of the wild-type −4200/+11600 LacZ transgene was relativelyweak in many SMC tissues until embryonic day (ED) 17.5-19.5. Thus, tofacilitate analysis of effects of CArG mutations during development, werestricted analyses to ED 19.5. Results showed the transgene expressionpattern in each CArG mutant transgenic mouse was largely consistent withthat in adult mice (FIG. 22). No expression was observed in embryos ofCArG1 mutant transgenic mice. In the CArG2 mutant transgenic mice,reporter expression was observed only in the GI tract. In intronic CArGmutant transgenic mice, the expression in the GI tract and airways wasequivalent to that in the wild-type transgenic lines. While reporterexpression in smaller arteries was easily detectable, no expression wasdetected in the large arteries in the intronic CArG mutant transgenicmouse embryos.

In summary, the transgenic mouse data demonstrate that each CArG elementis differentially required in SMC-subtypes in vivo in transgenic mice.CArG1 is crucial for transcription in all SMCs; CArG2 is indispensablein large blood vessels but had a relatively minor role in the GI andurinary tracts; the intronic CArG is absolutely required only in largeelastic arteries. Taken together, the results demonstrate themultiplicity of regulatory programs that control expression of SMCdifferentiation marker genes in vivo and indicate that each of themultiple CArG elements mediates distinct information for transcriptionalregulation in different cell-types in vivo. In addition, the dataindicate that the spatial and temporal regulation of SMC genes is notgoverned by a single regulatory region or an enhancer.

6. Discussions

The results of the transgenic mice of the CArG mutant constructsindicate that at least two regions (i.e., the 5′-flanking CArG andintronic CArG regions) are required for in vivo transcription of theSM-MHC gene. We are further mapping transcriptional regulatory modulesin the SM-MHC gene locus. Preliminary data indicate that the 5′-flankingand first intron contain multiple positive and negative transcriptionalregulatory regions, and that different SMC-subtypes require differentsubsets of modules (Manabe and Owens, unpublished observations). Whymight SM-MHC transcription require such a complex transcriptionalregulatory scheme in vivo?

It is evident that vascular SMCs within different vascular beds residein vastly divergent local environments in vivo. Differences in thephysiological role of vascular beds with respect to blood pressure,flow, and tone require very diverse vessel wall structures. SMCs arethus undoubtedly exposed to quite different vasoactive/neuronal stimuliand environmental cues from one vascular bed to another. If oneconsiders the differences between vascular and visceral SMCs, thediversity is even more prominent, and it is well established that SMCsare derived from different embryological origins. Finally, one must alsoconsider that the functions of SMCs can vary greatly during developmentand in adult animals due to their key role in matrix deposition, andvessel morphogenesis, as well as in vascular repair. Due to these manydifferences, SMCs in vivo need to respond to very diverse inputs(environmental cues) that activate various intracellular signalingpathways, and coordinately express necessary genes. It is thusconceivable that even to control the same gene, such as SM-MHC,SMC-subpopulations in different environments may need to utilizedistinct sets of regulatory pathways. In other words, the SM-MHC generegulatory program evolved so that it utilizes various regulatorypathways to control transcription in heterogenous extra-andintracellular environments. In fact, the differential requirement of theintronic CArG and CArG2 of the SM-MHC gene supports a hypothesis thatdistinct transcriptional regulatory programs are activated inSMC-subtypes.

One striking feature of the intronic CArG mutant was that the transgenewas completely silent in the elastic arteries such as the aorta, whereasexpression was easily detectable in the intermediate and small sizearteries directly branched from the aorta. There are at least twopossible explanations for the differential requirement of the intronicCArG that are not necessarily mutually exclusive. First, we need toconsider the heterogeneity in the embryonic origin of SMCs between thelarge and smaller arteries. It has been postulated that SMCs have atleast three embryonic origins: local mesenchymal cells, neural crestcells, and proepicardial cells. In the aorta, the aortic bulb andascending aorta mainly consist of neural crest derived SMCs and thedescending aorta mainly consists of mesenchymal derived SMCs. However,in intronic CArG mutant transgenic mice, SMCs in the aorta were stainednegative irrespective of the position of the cells, and no knowndifference in lineage fits the distribution of the intronicCArG-dependency. Therefore, it is unlikely that differences in embryonicorigin solely determined the requirement of intronic CArG fortranscriptional control.

In addition, as discussed above, the heterogeneity in phenotype andfunction of SMCs in vivo is likely to require multiple transcriptionalprograms to control the same gene. The differences in the physiologicalfunctions of SMCs in elastic versus muscular arteries would also requireSMCs to express distinct sets of genes to fulfill their functionalroles. It is thus conceivable that the intronic CArG is integrated in aregulatory program that processes environmental cues unique to elasticarteries and controls gene expression important for the function of suchvessels. A number of genes including ion channels, contractile proteins,growth factors/receptors, and transcription factors have been shown tobe differentially expressed in vascular beds. For example, atranscription factor, CHF1 (Hrt2/Hey2/HERP1/gridlock) has been shown tobe mainly expressed in the aorta. It would be of interest to compare thetranscriptional regulatory mechanisms of these genes, and also todetermine the function of differentially expressed transcription factorsin control of SMC-subtype-selective gene regulatory programs.

Example 5 SRF Interaction with SM-MHC CArG Elements

1. SMCs in Intact Tissues Expressed SRF and other Proteins that Bind toCArG Elements

As an initial step to determine mechanisms that controlSMC-subtype-specific transcriptional regulation through multiple CArGelements, we examined protein binding properties of each CArG elementusing EMSAs. Since there were no SMC culture cell lines that had beenshown to faithfully emulate differentiated phenotypes of SMC-subtypes interms of SM-MHC transcriptional control, we prepared nuclear extractsfrom intact rat tissues. As shown in FIG. 19, each CArG probe formedseveral DNA-protein complexes with tissue nuclear extracts. The mobilityof major shift bands (complexes A and B) formed with tissue nuclearextracts was the same as that with cultured SMC nuclear extracts. Themobility of complex A seen in tissues and culture cells was identical tothat formed with recombinant serum response factor (SRF). Supershiftassays using anti-SRF antibody showed that both complexes A and Bcontained SRF (FIG. 23). Several non-SRF shift bands that werespecifically competed by cold self-competitors but not by unrelatedsequences (data not shown) were also formed in the EMSA experiment(complexes C-G). Each probe formed largely similar shift band patternswith the SM tissue nuclear extracts. However, the shift bands formedwith liver or cultured SMC extracts were somewhat different from thoseformed with the SM tissues extracts. For example, CArG1, CArG2, andintronic CArG probes formed complex E with liver and cultured SMCextracts, while this complex was not formed with the SM tissue extracts.Conversely, complex F formed the intronic CArG probe and SM tissuesamples was not present in the liver samples. Although a furtheranalysis is necessary to determine the significance of these non-SRFDNA-binding proteins in transcriptional regulation in cells, the datashow that non-SRF DNA-binding proteins capable of binding to the CArGprobes may be differentially expressed in SM and non-SM tissues.

2. SRF Binding of CArG Elements of the SM-MHC Gene within IntactChromatin Under Physiological Conditions

To directly address whether SRF bound the endogenous SM-MHC CArGelements, we employed chromatin immunoprecipitation (ChIP) assays.Intact cultured rat aortic SMCs, L6 rat myoblasts, L6 myotubes, and Rat1fibroblasts were directly fixed with formaldehyde. Crosslinked chromatinwas immunoprecipitated with anti-SRF antibody. The precipitatedchromatin DNA was then purified and subjected to PCR analysis forenrichment of the target sequences. The promoters of insulin, β-globin,and skeletal α-actin genes (FIG. 24, Rows A-C), which are silent inSMCs, and a region (−4133 to −3832) of the SM-MHC 5′-flanking sequence(FIG. 24, Row D), which lacks CArG elements, were used in controlreactions. Amplification of these sequences showed a background level ofchromatin immunoprecipitation and PCR amplification (FIG. 24, Rows A-D).However, anti-SRF antibody specifically enriched the 5′-flanking CArGregion (CArG1 and CArG2) and the intronic CArG regions of the SM-MHCgene (FIG. 24, Rows E and F, lanes 3) in SMC chromatin as compared withthe background amplifications of the promoters of negative control genes(FIG. 24, Rows A-D, lanes 3). Importantly, the same SM-MHC regions werenot enriched in immunoprecipitation samples of L6 or Rat1 cells that donot express the SM-MHC gene (FIG. 24, Rows E and F, lanes 6, 9, 12).Since the promoter region of the skeletal α-actin has been shown tocontain CArG elements active in skeletal myocytes, this promoter wasused as a positive control for L6 cells. As expected, the skeletal actinpromoter was enriched in SRF immunoprecipitation samples from L6myoblasts and myotubes (FIG. 24, Row C, lanes 6 and 9) but not in SMC orRat1 cells, further demonstrating the specificity of the SRF antibody inthese experiments. It is important to note that the PCR detectionmethods used in these ChIP experiments are not quantitative. As such, itis impossible to determine the stoichiometry of SRF binding to theSM-MHC CArG elements. Nevertheless, the ChIP experiments indicated thatat least some SM-MHC CArG regions were bound by SRF in chromatin inintact cultured SMCs. In addition, observations that SRF bound the CArGregions of endogenous SM-MHC gene in chromatin in intact SMCs but not inL6 skeletal myocytes or Rat1 fibroblasts provide evidence thatmechanisms exist in vivo to control SRF binding to the SM-MHC CArGs in acells-specific manner.

3. Discussion

SRF-dependent control of SM-MHC transcription in vivo: The presentstudies provide evidence showing binding of SRF to the CArG elements ofthe endogenous SM-MHC gene in the context of intact chromatin as opposedto oligonucleotide fragments employed in typical DNA binding studies. Ithas been shown that in an in vitro avian proepicardial celldifferentiation system two types of dominant negative SRF inhibited SMCdifferentiation and reduced expression of SMC marker genes including SMα-actin and SM22α. These data demonstrate the significant role of SRF inthe control of endogenous SMC differentiation marker genes. A criticalquestion is thus: How can SRF, which is not clearly SMC-specific,regulate SMC-specific gene expression? Various hypotheses have beenpostulated that are not mutually exclusive. First, although SRF isclearly not cell specific, there are very large differences in the levelof SRF expression between different cell types that may contribute, atleast in part, to cell specific SRF-CArG dependent gene expression.Second, the binding affinity of SRF may be regulated in a cell typedependent manner by interactions with other proteins, such as MHox, orby phosphorylation. Third, SRF may form SMC-specific multi-proteincomplexes. Although we did not observe SMC-specific higher ordercomplexes in EMSA experiments, it is possible that a longer probe mightform such a multi-protein complex in EMSAs. Lastly, chromatin remodelingmay play a significant role in the regulation of activity oftranscriptional regulatory modules. It is now well established thattranscription factor binding to cis-elements are greatly affected bychromatin structure. It has also been shown that various transcriptionfactors bind histone acetylases and deacetylases and thereby modifychromatin structure.

The results of ChIP assays (FIG. 24) demonstrated that the SM-MHC CArGregions were bound by SRF only in SMCs, although nuclear extracts of L6myocytes were perfectly capable of binding the CArG elements in EMSAs(Manabe and Owens, unpublished observations). Conversely, SRF bound theskeletal α-actin promoter only in L6 myocytes but not in SMCs orfibroblasts. These data are potentially extremely important in that theysuggest that the transcriptional regulatory regions of the endogenousSM-MHC gene are only active in SMC chromatin. That is, thetranscriptional regulatory regions of the SM-MHC gene may be in “closedstate” in the non-SM cell lines.

SMC-subtype-selective transcription control in vascular diseases: Incontrast to the main function of mature SMCs (i.e., contraction), one ofthe major functions of SMCs in developing blood vessels is to contributeto formation of the vascular wall through cell proliferation andproduction of extracellular matrix components. Such functions are alsoextremely important during repair of vascular injury, and may contributeto post-angioplasty restenosis. As such, it is likely that a part of thetranscriptional regulatory programs that are normally activated invascular development is re-activated by vascular injury and alters geneexpression. It would be thus important to study the functions of theCArG elements during vascular development and in neointimal formationinduced by vascular injury.

It is well known that some vascular beds including the coronary arteriesand aorta are more prone to atherogenesis. Our data provide evidence forSMC-subtype-selective transcriptional regulatory mechanisms. It istempting to speculate that this multiplicity in the transcriptionalcontrol mechanisms might in some way be related to differentialsusceptibility of different vessels to atherosclerosis. The modularityof the SM-MHC transcription program might also allow us to design genetherapy vectors to target specific subsets of SMCs. SMC-selectiveactivity obtained by the intronic CArG region coupled with a minimal TKpromoter in transgenic mice suggests that the region could be used as abuilding block for such vectors. The results of the present studies haverevealed the complex nature of transcriptional control of the SM-MHCgene in vivo in SMC-subtypes, and the role that multiple cis-regulatorymodules play in processing divergent environmental cues in vivo. Furtherstudies on the SM-MHC gene regulation should provide additional insightsinto the complex and dynamic regulatory mechanisms that normally controlSMC differentiation and how these processes are altered duringphenotypic modulation of SMCs during injury repair and development ofvascular diseases.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

1. An isolated polynucleotide comprising a smooth muscle cell myosinheavy chain (SM-MHC) promoter/enhancer, wherein the enhancer comprisesnucleotides 5638-5860 of SEQ ID NO:16 or nucleotides 6862-7100 of SEQ IDNO:17, and the promoter comprises a heterologous TATA box ortranscription initiation site, and wherein the promoter/enhancerinitiates expression in a smooth muscle cell in vivo when introducedinto an animal.
 2. The polynucleotide of claim 1, wherein the promotercomprises a CArG1 and/or a CArG2 motif.
 3. The polynucleotide of claim1, wherein the enhancer is coupled to a minimal thymidine kinase (TK)promoter.
 4. The polynucleotide of claim 1, wherein the promoter isoperably linked to a heterologous polynucleotide.
 5. The polynucleotideof claim 4, wherein the heterologous polynucleotide encodes apolypeptide.
 6. An isolated polynucleotide comprising a smooth musclecell myosin heavy chain (SM-MHC) promoter/enhancer, wherein thepromoter/enhancer sequence comprises SEQ ID NO:16 or SEQ ID NO:17,wherein: a CArG2 motif is mutated and the promoter is expressed inmesenteric artery, airway, stomach, intestine and bladder but not aorta,coronary artery or vena cava when introduced into an animal; or theintronic CArG motif at positions 5815-5825 of SEQ ID NO:16 or 7046-7056of SEQ ID NO:17 is mutated and the promoter is expressed in coronaryartery, mesenteric artery, vena cava, airway, stomach, intestine andbladder but not aorta when introduced into an animal.
 7. Thepolynucleotide of claim 6, wherein the promoter/enhancer comprises SEQID NO:16 and the CArG2 motif is mutated.
 8. The polynucleotide of claim6, wherein the promoter/enhancer comprises SEQ ID NO:16 and the intronicCArG motif is mutated.
 9. The polynucleotide of claim 6, wherein thepromoter is operably linked to a heterologous polynucleotide.
 10. Thepolynucleotide of claim 9, wherein the heterologous polynucleotideencodes a polypeptide.
 11. An isolated genetically engineered cellcomprising the polynucleotide of claim 1 or
 6. 12. A compositioncomprising the polynucleotide of claim 1 or 6 in a pharmaceuticallyacceptable carrier.
 13. An isolated polynucleotide comprising a smoothmuscle cell myosin heavy chain (SM-MHC) promoter/enhancer, wherein thepromoter/enhancer sequence comprises: nucleotides 1 to 9500 and 11,700to 13,700 of SEQ ID NO:16 and does not comprise the interveningnucleotides 9501-11699 of SEQ ID NO: 16; or nucleotides 1 to 6700 and9,500 to 15,800 of SEQ ID NO:16 and does not comprise the interveningnucleotides 6701-9499 of SEQ ID NO:16; and wherein the promoter/enhancercomprises a mutated or unmutated CArG2 or intronic CArG motif and thepromoter/enhancer initiates expression in pulmonary vascular and airwaysmooth muscle cells in vivo when introduced into an animal.
 14. Theisolated polynucleotide of claim 13, wherein the promoter/enhancercomprises nucleotides 1 to 9500 and 11,700 to 13,700 of SEQ ID NO:16 anddoes not comprise the intervening nucleotides 9501-11699 of SEQ IDNO:16.
 15. The isolated polynucleotide of claim 13, wherein thepromoter/enhancer comprises nucleotides 1 to 6700 and 9,500 to 15,800 ofSEQ ID NO:16 and does not comprise the intervening nucleotides 6701-9499of SEQ ID NO:16.
 16. The isolated polynucleotide of claim 13, whereinthe promoter/enhancer initiates expression in gastrointestinal, airway,arteriolar, and bladder smooth muscle cells but does not initiateexpression in vascular smooth muscle cells within large arteries. 17.The isolated polynucleotide of claim 13, wherein the promoter/enhancercomprises a mutated CArG2 motif.
 18. The isolated polynucleotide ofclaim 13, wherein the promoter/enhancer comprises an unmutated CArG2motif.
 19. The isolated polynucleotide of claim 13, wherein thepromoter/enhancer comprises a mutated intronic CArG motif.
 20. Theisolated polynucleotide of claim 13, wherein the promoter/enhancercomprises an unmutated intronic CArG motif.
 21. The isolatedpolynucleotide of claim 19, wherein the promoter/enhancer initiatesselective expression in vascular smooth muscle in arterioles and airwaysmooth muscle.
 22. The isolated polynucleotide of claim 17, wherein thepromoter/enhancer initiates selective expression in gastrointestinalsmooth muscle.
 23. An isolated genetically engineered cell comprisingthe polynucleotide of claim
 13. 24. A composition comprising thepolynucleotide of claim 13 in a pharmaceutically acceptable carrier. 25.The polynucleotide of claim 1, wherein the enhancer comprisesnucleotides 5638-5860 of SEQ ID NO:16.
 26. The polynucleotide of claim1, wherein the enhancer comprises nucleotides 6862-7100 of SEQ ID NO:17.27. The polynucleotide of claim 6, wherein the promoter/enhancercomprises SEQ ID NO:17 and the CArG2 motif is mutated.
 28. Thepolynucleotide of claim 6, wherein the promoter/enhancer comprises SEQID NO:17 and the intronic CArG motif is mutated.